Preclinical study of Meso CAR-T for the treatment of solid tumors

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Preclinical study of Meso CAR-T for the treatment of solid tumors

Preclinical study of Meso CAR-T for the treatment of solid tumors.

Brief Introduce

Mesothelin is a glycosyl-phosphatidylinositol-linked membrane glycoprotein, which is expressed in many cancers.

Immunohistochemical studies have shown that mesothelin is overexpressed in almost all pancreatic ductal adenocarcinoma and mesothelioma, as well as a high proportion of epithelial ovarian cancer and non-small cell lung cancer .

Since it has recently been reported that mesothelin binds to CA125/MUC16, a possible biological effect of this membrane protein may be related to atypical cell adhesion, which may promote the metastasis and spread of mesothelin-carrying cancer cells.

In early studies, tumor cells expressing mesothelin have been shown to be killed by MHC class I restricted T cells.

In addition, in preclinical and clinical studies, mesothelin-specific antibodies and antibody-based immunotoxins have been shown to cause tumor regression.

Here we report that lentiviral vectors can effectively generate T cells specifically targeting mesothelin.

Mesothelin-retargeted T cells eradicate NOD/scid/IL2rγ–/– large pre-established mesothelioma xenografts in mice with a low effector to target (E:T) ratio, and costimulatory domains The combination can prolong the administration of T cells in tumor-bearing mice within the engineered persistent tumor or after intravenous injection.

Test results

The chimeric receptor was designed to contain SS1 scFv that recognizes human mesothelin (Figure 1A).

SS1 was chosen because of its high binding affinity for mesothelin (Kd=0.7 nM), and it was found to be safe for patients when administered as a recombinant immunotoxin (13).

We have created a series of chimeric receptors based on SS1 scFv, which contain the TCR-ζ signal transduction domain and the tandem CD28 and CD137 (4-1BB) intracellular domains.

Chimeric receptors containing truncated forms of TCR-ζ intracellular domain (SS1-Δz) or anti-CD19 scFv (anti-CD19-z) were designed as controls for signal transduction and binding specificity, respectively.

Preclinical study of Meso CAR-T for the treatment of solid tumors.

Figure 1 Generation and cytolytic activity of engineered T cells with anti-mesothelin lentiviral vector .

We use lentiviral vector technology to express the fusion construct in primary human T cells using clinically validated technology.

The cDNA sequences containing various fusion constructs were cloned into the third-generation lentiviral vector, in which the CMV promoter was replaced by the EF-1α promoter.

The supernatant of the lentiviral vector efficiently transduced primary T cells (Figure 1B).

In preliminary experiments, it was found that SS1 transduced T cells continued to proliferate in the presence of various mesothelin-expressing cell lines, and cultured in the absence of mesothelin-expressing feeder cells failed to maintain T cells. proliferation.

All constructs are brightly expressed on the surface of CD4 and CD8 T cells, and the expression is stable for at least 2 months in culture.

Compared with T cells expressing only the SS1 TCR-ζ signal domain, T cells containing SS1 scFv fused with any costimulatory domain have higher T cell expansion during culture with mesothelin-expressing tumor cells.

This is consistent with previous reports that T cells and other chimeric receptors have been tested in vitro.

In addition, during culture on mesothelin-expressing tumors, the SS1 scFv fused to the costimulatory domain enriches the T cell population to near purity.

In order to study the anti-tumor potential of transduced T cells, mesothelin-negative K562 cells, K562.meso (a derivative engineered to express mesothelin) and ovarian cancer patients and malignant mesothelioma were used (Figure 1C). ).

T cells transduced with SS1 scFv effectively lysed K562.meso, but did not kill the parental K562 cells. Importantly, SS1 transduced T cells are also highly cytotoxic to mesothelin-expressing cancer cells, and can kill OvCa68.4 and M108 cell lines derived from patients with ovarian cancer and mesothelioma, but cannot kill at the same time.

Ovarian tumor cells that do not express mesothelin (OvCa61.4).

Transduction of OvCa61.4 cells with a lentiviral vector expressing mesothelin makes them susceptible to SS1 scFv lysis.

Including CD28 and CD137 costimulatory domains in tandem or in triplicate with TCR-ζ usually cannot increase in vitro cytotoxicity to T cells expressing only SS1-TCR-ζ.

Killing is effective. Platform lysis occurs at a 10:1 E:T ratio during the 4-hour measurement period and <1:1 E:T ratio during the 48-hour culture period [Figure S1], indicating that the redirected T cells can Continuous killing. In addition, lysis is specific because T cells transduced with GFP or an unrelated anti-CD19 scFv chimeric receptor have no cytotoxic activity against the same target cells, excluding alloreactivity or non-specific lysis. In addition, T cells expressing the truncated TCR-ζ intracellular domain (SS1-Δz) also failed to kill mesothelin-expressing targets, indicating that a complete TCR-ζ signal domain is required.

Studies on mouse tumor models have shown that the anti-tumor effects of redesigned CD8 T cells may be maintained by CD4 cells.

Therefore, we transduced primary human CD4 and CD8 T cells and measured cytokine secretion after exposure to mesothelioma cells (Figure S2A).

The release of inflammatory cytokines is triggered by mesothelin, and both CD4 and CD8 T cells secrete Th1 cytokines.

In contrast, T cells secrete small or undetectable amounts of IL-4, IL-5, IL-10, and IL-17, which is consistent with the major Th1 cytokine bias under these culture conditions.

The observed cytokine release patterns are consistent with the known signal transduction properties of various signal transduction domains, confirming the modular nature of the domains and their ability to act on fusion proteins in cis.

For example, IL-2 and TNF-α are only secreted by CD4 T cells, depending on the CD28 domain. Both CD4 and CD8 T cells release IFN-γ and IL-6, and the CD28 or CD137 signal domain is sufficient.

Therefore, in contrast to cytotoxicity, when TCR-ζ is sufficient, cytokine secretion is more pronounced in primary T cells expressing costimulatory signal domains.

No mesothelin-independent cytokine secretion was observed.

An emerging concept in the field of vaccines suggests that the presence of multifunctional T cells is related to improving the control of chronic viral infections, which may be important for T cell-based cancer therapies.

The transduced T cells were stimulated with tumor cells expressing mesothelin, and the subpopulations of CD4 and CD8 T cells stained intracellular IFN-γ, TNF-α, IL-2 and GM-CSF (Figure S2B).

The production of cytokines is limited to the portion of T cells that express the chimeric receptor.

The incorporation of the CD28 signal domain increased the proportion of cytokine secreting CD4 and CD8 T cells in the early stage of culture; however, after 20 hours of culture, the proportion of cytokine secreting cells in response to all signaling constructs was similar.

However, the proportion of multifunctional T cells is highly dependent on the presence of costimulatory domains, because almost all cytokine secreting cells that only express TCR-ζ domains are monofunctional.

In contrast, highly multifunctional T cells that secrete all four cytokines are mainly observed in cells expressing the BB:TCR-ζ or 28:BB:TCR-ζ signal domain.

Importantly, the CD137 signal domain is known for supporting multifunctional CD4 and CD8 T cells, and this feature may extend the function of T cells in the tumor microenvironment.

The expression of anti-apoptotic genes can confer resistance to the toxic effects of the tumor microenvironment.

Most T cells that express CD28, and to a lesser extent the CD28:BB signal domain, express Bcl-XL after being cultured with the M108 mesothelioma cell line (Figure S2C).

In contrast, T cells expressing BB:TCR-ζ or truncated TCR-ζ have low or undetectable levels of Bcl-XL.

The expression of Bcl-XL in T cells expressing the CD28 and CD137 signal domains is not constitutive, because T cells cultured in medium (see Figure S2C) or mesothelin-negative tumor cells do not express detectable levels of Bcl -XL.

To further explore the potential anti-tumor efficacy of the SS1 scFv construct, we developed a strict xenograft model using M108 tumor cells.

When implanted into the flanks of NOD/scid/IL2r γ– / – (NOG) mice, M108 cells proliferated and continued to express mesothelin (Figure S4 ).

Serially passaged primary M108 tumor cells were injected into the flanks of NOG mice in each group (Figure 2A ).

After the establishment of the tumor . 6 weeks and . 7 weeks, when tumor burden ≈ mm3 500 , the mice received an intratumoral injection of 15 × 106 T cells (≈ 70% – 80% therapeutic transgene positive).

An effective anti-tumor effect was observed (see Figure 2A ). Statistical models of tumor growth curves revealed a significant difference between treatment groups ( P <from 0.0001 by Wald All groups were given the same tumor volume logarithmic curve test results) .

7 curves ared divided into 4 groups: The 3 control treatments (saline, GFP and Δ z ) have no significant difference between each other, and they still show continuous growth after T cell transfer;
TCR- ζ treatment shows in some animals The mixed mode of continuous growth, but slowed down growth or tumor decline in other animals (Figure 2B );
BBz treatment showed relatively slow tumor regression;
28z and 28BBz treatments showed rapid tumor regression, and it was statistically impossible distinguish.

Figure 2 Mesothelin retargets T cells to eradicate large pre-established tumors in vivo: the effect of costimulatory signal domain and route of administration.

The tumor burden map of a single mouse (see Figure 2B) shows the size and reproducibility of the anti-tumor effect of T cells with chimeric receptors containing costimulatory domains.

Among them, one mouse was palpable after the tumor regressed s level. Kill the mouse when the tumor volume is> 2,000 mm3, and take pictures of the tumor (Figure S5).

The tumors of mice treated with control T cells were highly vascularized, while many small tissue pieces harvested from 28z or 28BBz mice and some BBz mice were fibrotic or necrotic pieces.

However, according to histological determination, small tumor areas with abnormal morphology are still obvious.

Therefore, T cells expressing mesothelin-specific chimeric receptors containing CD28 and 4-1BB domains can control or eradicate large tumors that have formed after intratumoral injection.

The allogeneic response did not significantly contribute to the anti-tumor effect, because the tumor growth of mice treated with T cells expressing GFP or truncated TCR-ζ receptor was the same as that of mice injected with saline.

In previous experiments, strong and specific anti-tumor effects were observed in mice treated by intratumoral injection of engineered T cells, and their potential anti-tumor effects in the vascularized tumor microenvironment were directly evaluated.

We next tested whether T cells would be effective using multiple routes of administration (Figure 2C). Use M108 cells to establish tumors.

After the tumor reaches an average volume of ≈500 mm3, in the 6th and 7th weeks, 10×106 T cells transduced with SS1-28:BB:TCR-ζ (>90% positive) are injected .

It is given by iv, ip or intratumoral route. For these experiments, the CD28:BB:TCR-ζ construct was chosen because it shows the most effective and reproducible anti-tumor effect, as well as enhanced implantation properties (see below).

Following intravenous injection and intratumoral injection, effective anti-tumor effects were again observed, and the tumor mass decreased more rapidly after intratumoral administration.

We analyzed the tumor volume on a natural logarithmic scale. For the logarithmic tumor volume, the ranking (from low to high) is intratumoral SS1-28BBz, intravenous injection. SS1-28BBz, IP SS1-28BBz, intratumoral anti-CD19-28BBz and normal saline.

The overall F test used to compare means is significant at P <0.0001. In pairwise comparisons, each treatment is not significantly different from adjacent treatments (P = 0.05), but is significantly different from other treatments.

Therefore, we concluded that the eradication of M108 tumors by SS1-28BBz is antigen-specific, because the anti-CD19-28BBz group did not show anti-tumor activity, and intratumoral seems to be a better route of administration, slightly faster than intravenous injection. (≈7 days response delay) but obviously better than ip.

Because a surprising short-term implantation was observed in pilot clinical trials to test chimeric receptors , and because long-term implantation of adoptively transferred T cells is associated with anti-tumor effects , we next determined that in tumor-bearing mice Lentiviral vector engineered T cell.

We first examined the mice in the experiment shown in Figure 2A . On day 73 (ie 20 days after the last adoptive T cell transfer ) , peripheral blood of M108 NOG- bearing mice that received intratumoral injection of SS1 scFv -transduced T cell therapy on day 46 and day 53 after tumor injection were obtained , And quantify the presence of CD4 and CD8 T cells (see Figure 3A ).

For all test conditions, the CD8 T cell count is higher than the CD4 T cell count. The analysis of variance of the CD4 data showed that there were significant differences between the treatment groups ( P <0.0001 ).

In the pairwise comparison of treatment groups, passTukey method of adjusting multiplicity, imparting 28BBz cell counts in mice was significantly higher than BBZ and the GFP , and BBZ , 28Z , the Zeta and [Delta] Z group can not be distinguished.

The analysis of CD8 data also revealed significant differences between the treatment groups ( P <0.0001 ).

In Tukey pairwise comparisons adjusted in transplanted expression 28BBz and BBZ cell counts in mice cells significantly higher (P <0.05) the GFP , and 28BBz , BBZ , the Zeta , 28Z , and [Delta] Z group can not be distinguished.

Unexpectedly , the T cell count in mice given 28z -transduced T cells was not higher than that of only expressing TCR- [zeta] of the T cells, because they have an excellent IL-2 production and increased Bcl-XL expression, as shown in S2 ) .

Furthermore, peripheral blood T persistence cell tumor burden no direct correlation (FIG 2A Compare and B with Figure 3A ).

Preclinical study of Meso CAR-T for the treatment of solid tumors.

Figure 3 CD28 and 4-1BB signals enhance the persistence of human T lymphocytes after M108 tumor treatment.

Finally, we measured the persistence of T cells in mice given 28BBz as a function of the route of administration.

In the experiment shown in Figure 2C, mice were injected with T cells on day 43 and day 49, and blood was taken on day 65 to measure T cell transplantation.

The tumor volume and peripheral blood persistence of each mouse in each group on day 65 are plotted (see Figure 3B).

By checking the drawing, the presence of SS1-28:BB:z T cells is usually inversely proportional to the tumor burden, because the group of mice injected with T cells intratumorally or intravenously.

All routes have a large number of T cells and the lowest tumor burden in the peripheral blood, while mice given by intraperitoneal injection of SS1-28:BB:z or intratumoral administration of anti-CD19-28:BB:z have high Tumor burden and low-level T cell transplantation.

The overall F test comparing the average T cell counts of each group was significant (P<0.0001).

The group with the highest average T cell count was intravenous injection.

In the pair-wise comparison of means adjusted by Tukey, the count of iv SS1 administration was significantly higher than that of ip (P<0.05).

SS1 administration and intratumoral anti-CD19 scFv administration.

The T cell count of mice treated with intratumoral SS1 was also significantly higher (P<0.05) than in the intratumoral anti-CD19 group, indicating that tumor antigens drive the expansion of adoptively transferred T cells in vivo.

In addition, except for low-level T cell transplantation, the tumor burden of mice injected with T cells expressing non-binding anti-CD19 scFv 28:BB:z receptors was equivalent to that of mice injected with saline, indicating the specificity of T Triggering the high-level transplantation and anti-tumor effects observed for T cells expressing SS1 scFv 28:BB:z receptors requires M108 cells to affect the cells, not allogeneic or xenogeneic effects.

Therefore, the long-term systemic persistence of SS1 scFv 28:BB:z engineered T cells is driven by tumor antigens and occurs in tumor-bearing mice intratumorally or after intravenous injection.

Statistically, intravenous injection is equivalent to intratumoral injection, but it is significantly better than intraperitoneal injection.

Discussion

Although the initial clinical studies proved the feasibility of retargeting T cells, the persistence and poor expression of the transgene in the body have been documented, and the clinical activity of the therapy is lower than expected (2-4) .

Our research has solved all these problems. Mesothelin proved to be an attractive target because the retargeted T cells effectively and specifically killed various tumors expressing mesothelin.

The modified T cells can kill tumor cells in vitro with an E:T ratio of about 1:10 , and in vivo with an unprecedented 1:40 E:T ratio.

Unexpectedly, the expression of the CD137 signaling domain is most correlated with the persistence of T cells in vivo , because the advantages of in vitro measurements of cytokine function and Bcl-XL expression predict that the CD28 domain is the most effective.

We provide evidence that T cells expressing SS1 fused with CD28 and CD137 costimulatory domains can reject very large, mature tumors of 500 to 1,000 mm3.

Given that the experiment in Figure 2A used 2 T cell injections, containing a total of 20 to 24 × 106 T cells, these T cells were transduced with the SS1 construct, and assuming that the 1,000-mm3 tumor mass contains at least 1 × 109 cells, We estimate that T cells can eradicate tumors in vivo with an initial E:T ratio of ≈1:40.

Therefore, our preclinical data will support the treatment of patients with a tumor burden of at least 1 × 10 12 cells, because our current large-scale production can routinely produce 1 × 10 11 transduced T cells during a 10-day culture process .

There may be several mechanisms for the increased efficiency of redirecting T cells observed in this study. First, previous studies usually used T cells transduced with retroviruses.

The high efficiency associated with lentivirus-mediated transduction, combined with powerful in vitro cell expansion methods, makes rapid expansion of a large number of therapeutically relevant quantities feasible.

In this study, we used a lentiviral vector with higher transduction efficiency, thus shortening the in vitro culture time to 7 to 10 days, and allowed us to show before that the length of the average telomere cultured T cell is actually longer than that of the culture Start time.

We attribute this to previous evidence that the anti-CD28-driven culture system induces telomerase activity and retains central memory cells.

It may be important to use T cells with longer telomeres, because these cells have a broader ability to replicate, and in adoptive transfer studies of tumor-infiltrating lymphocytes, it has an enhanced effect on patients injected with “young” tumor-infiltrating lymphocytes.

Anti-tumor effect. Secondly, compared with previous reports, our transgene is expressed brightly on the surface of T cells.

This may be the result of lentiviral vector design, the use of EF-1α as an internal promoter, and the use of CD28 co-stimulation to increase the expression level of the transgene.

Third, we show that the binding of costimulatory domains in cis enhances the persistence of T cells, confirming and extending recent work, showing that costimulatory domains expressed in trans in T cells can enhance retargeting of T cells The effect of (38).

Our research shows that in vitro experiments can be misleading in predicting the efficacy of engineered T cells in vivo.

In addition, engineered cells expressing the CD137 signal domain are more likely to be multifunctional and persist in tumor-bearing mice.

We tested several routes of administration and found that the intratumoral and intravenous routes were almost equivalent and significantly better than the ip route. Intravenous injection delays tumor regression.

Compared with intratumoral route, the soluble mesothelin in mouse plasma can be at least partially explained, which reflects that mesothelin-expressing tumor patients have been shown to have circulating mesothelin.

We hope to mainly test the approach in iv Phase I clinical studies; however, the intratumoral approach may have certain advantages. First, the transport of T cells into solid tumors may be rate-limiting, and direct injection eliminates transport as a variable.

Second, in the presence of potential intra-target and extra-organ toxicity, the intratumoral route may increase the therapeutic index.

Finally, recent studies have shown the safety and feasibility of intratumoral injection of T cells. In summary, chimeric T cells that target mesothelin and have intracytoplasmic signaling domains from TCRζ, CD28, and CD137 appear to be very effective in treating large, mature tumors in mice.

The clinical transformation of this approach to tumors, such as mesothelioma and ovarian cancer, is currently underway.

Reference:

PNAS:orgcgidoi10.1073pnas.0813101106

Preclinical study of Meso CAR-T for the treatment of solid tumors.

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