TW202304512A - Dosing of bispecific t cell engager - Google Patents

Abstract

Methods for reducing myeloid-derived suppressor cells and activating T cells in a patient and for treating a patient suffering from a solid tumor are described. The methods entail administering a CD3/CD33 T cell engager.

Description

Administration of bispecific T cell engagers

T cell adapters are a class of specific bispecific antibodies that mediate the binding between target cells and T cells, causing directed lysis of T cells and activation, differentiation and proliferation of T cells. While T cell engagers have demonstrated impressive potency and anti-tumor activity in some instances, in many instances an obstacle to broader therapeutic success has been undesirable activity on normal cells expressing the target of interest. This "on-target, off-tumor" toxicity can be significant and has been widely reported for engineering T cell engagers.

Myeloid-derived suppressor cells (MDSCs) act locally and systemically to impair antitumor immunity, suppress effector T cell responses, promote the formation of immunosuppressive regulatory T cells, inhibit maturation and antigen presentation of dendritic cells, and promote metastases form. MDSCs trigger a cascade of inhibitory functions that suppress normal cellular responses and result in unresponsiveness to immune checkpoint blockade. A major function of MDSCs is to suppress T cell activity in a variety of ways depending on the pathology and circumstances. The presence of MDSCs is thought to be associated with poor outcome and lack of response to certain therapies, such as those that activate T cells and those involving the use of checkpoint inhibitors.

Some T cell activation therapies (eg immunotherapies such as T cell engagers and CAR T cell therapy) are associated with cytokine release syndrome (CRS). The occurrence of CRS can limit the efficacy of some immunotherapies. T cell activation drives myeloid cell activation and the production of various cytokines and chemokines, including IL-6. In some instances, the levels of cytokines and chemokines are pathological. MDSCs are bone marrow cells that mainly produce IL-6.

Described herein are methods using AMV564, a bispecific, bivalent molecule that binds to CD3 and CD33. AMV564 is a homodimeric protein (that is, a homodimer of a polypeptide having the amino acid sequence of SEQ ID NO: 1), which has four single-chain variable fragment (scFv) binding sites, two of which bind CD33 and both bind CD3. In theory, a bivalent design could restore selectivity for T cell adapters, directing preferential binding to regions of high local density, such as those found at active signaling sites or associated with high receptor density or expression. Despite the fact that AMV564 binds CD33, which is broadly expressed throughout the myeloid lineage, it can be administered in a manner that provides a desirable therapeutic index and binds selectively to MDSCs. Importantly, AMV564 has been found to be selective over a broad dose range. Without being bound by any particular theory, this may be due to some combination of bivalency, affinity of the scFv and geometry of the homodimer. For example, also without being bound by any particular theory, the structure of AMV564 may allow it to bind clusters of dimeric CD33.

AMV564 has dual activity: it induces T cell-mediated MDSC killing and drives T cell activation, promoting favorable polarization (eg, Th1 CD4 T cells and effector CD8 T cells). The EC50 of AMV564 against MDSC is lower than about 3 pM. When administered appropriately, AMV564 largely avoids neutrophils, monocytes, and many differentiated myeloid cells while directing MDSC killing, thereby inhibiting MDSC-driven inhibitory pathways.

AMV564 is indicated for the reduction of MDSCs and can be used to reduce systemic immunosuppression, for example in patients with solid tumors. AMV564 can also be used in combination with various immunotherapies to control CRS and reduce immunosuppression.

Peripheral MDSCs can play an important role in T cell suppression and migration of T cells to tumor sites, a potential rate-limiting factor for T cell activation-based therapies. For example, in some cases, a dose of 15 µg of AMV564 can achieve significant depletion of MDSC populations. Since MDSCs are recruited from the bone marrow, peripheral depletion may allow sufficient control over the time frame of administration beyond the limited lifespan of tissue-resident MDSCs to favor anti-tumor immunity. However, the distribution of AMV564 in the tumor microenvironment can target MDSCs at the tumor site while promoting local T cell expansion. Furthermore, delivery of AMV564 to or into draining lymph nodes plus the periphery (e.g., at doses up to 50 µg, 75 µg, and 100 µg by the CIV route) achieved anti-tumor T cell desuppression and restored antigen presentation and immune homeostasis. Subcutaneous delivery of AMV564, eg, at doses of 5 µg, 15 µg or 50 µg, provides a direct mechanism for initial distribution in the lymphatic system, including tumor-draining lymph nodes. When administered subcutaneously, AMV564 was effective at lower doses, most likely due to entry into the lymphatic system.

AMV564 alleviates immunosuppression and activates T cell effector functions in cancer patients. AMV564 can achieve this by alleviating immunosuppression through targeted depletion of myeloid-derived suppressor cells (MDSCs) and by directly activating/repolarizing T cells and improving T effector function.

Importantly, subcutaneous administration of AMV564 promotes immune activation by targeting the lymphatic system.

Described herein are methods for reducing myeloid-derived suppressor cells and activating T cells in a patient (e.g., a patient undergoing immunotherapy), the method comprising administering to the patient AMV564 (having the amino acid sequence of SEQ ID NO: 1 of polypeptides). In various embodiments: AMV564 is administered by subcutaneous injection; the dose of AMV564 injected is 5 μg to 150 μg (micrograms); AMV564 is administered for at least 7 days (8, 9, 10, 11, 12, 13, or 14 days); daily subcutaneous administration of AMV564 (e.g., at 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μg/dose); 10 days over a 14-day period With AMV564; continuous administration of AMV564 for 5 days in two periods within 14 days; continuous administration of AMV564 for 5 days, no administration for the next two days, and subsequent continuous administration for 5 days; administration in a 21-day cycle AMV564, wherein AMV564 is administered for at least 7 days within a 14-day period and not administered during the subsequent 7-day period; the 21-day cycle is repeated at least twice; AMV564 is administered for at least 10 days within a 14-day period, wherein it is administered consecutively 5 days, no administration for the next 2 days, and then continuous administration for 5 days; the daily dosage of AMV564 administered at the time of administration was 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μg; The patient is treated with a therapy that activates T cells (e.g., the therapy is CAR T cell therapy; the therapy is CTL therapy; the therapy is antibody therapy; the therapy is treatment with a T cell engager comprising a CD3 binding domain that activates T cells); The patient has leukemia (acute myeloid leukemia or myelodysplastic syndrome) or is being treated for leukemia; the patient has or is being treated for a solid tumor; the solid tumor is selected from the group consisting of: pancreatic cancer, ovarian cancer, colorectal cancer , rectal cancer, non-small cell lung cancer, urothelial carcinoma, squamous cell carcinoma, rectal cancer, penile cancer, endometrial cancer, small bowel cancer, appendix cancer; administration of AMV564 achieves 0.1 pM to 5 pM (eg, 0.1, Steady-state exposure to AMV564 at 0.2, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0 pm).

Also described is a method for treating a solid tumor in a patient, the method comprising administering AMV564 (polypeptide having the amino acid sequence of SEQ ID NO: 1) to the patient. In various embodiments: AMV564 is administered by subcutaneous injection; the dose of injected AMV564 is 5 μg to 150 μg (mcg or micrograms); at least 7 days (8, 9, 10, 11, 12, 13, or 14 days) administration of AMV564; daily subcutaneous administration of AMV564 (e.g., at 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μg/dose); Administration of AMV564 for 10 days; continuous administration of AMV564 for 5 days in two periods within 14 days; continuous administration of AMV564 for 5 days, no administration for the next two days, and subsequent continuous administration for 5 days; in a 21-day cycle Internal administration of AMV564, wherein AMV564 is administered for at least 7 days within a 14-day period and not administered within the subsequent 7-day period; the 21-day cycle is repeated at least twice; AMV564 is administered for at least 10 days within a 14-day period, wherein Continuous administration for 5 days, no administration for the next 2 days, and continuous administration for 5 days; the daily dose of AMV564 administered at the time of administration was 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μg ; the patient is being treated with a therapy that activates T cells (e.g., the therapy is a CAR T cell therapy; the therapy is a CTL therapy; the therapy is an antibody therapy; the therapy uses a T cell engager comprising a CD3 binding domain and an activated T cell treatment); the patient has or is being treated for leukemia (acute myeloid leukemia or myelodysplastic syndrome); the patient has or is being treated for a solid tumor; the solid tumor is selected from the group consisting of: pancreatic cancer, ovarian Carcinoma, colorectal cancer, rectal cancer, non-small cell lung cancer, urothelial carcinoma, squamous cell carcinoma, rectal cancer, penile cancer, endometrial cancer, small bowel cancer, appendix cancer; administration of AMV564 achieved 0.1 pM to 5 pM AMV564 The steady-state exposure; Solid tumors are selected from the group consisting of: small cell lung cancer (NSCLC) (eg, metastatic nonsquamous NSCLC, III NSCLC, metastatic NSCLC expressing PD-L1), melanoma, Merkel cell carcinoma , MSI-high cancer (e.g., unresectable or metastatic, MSI-H, or mismatch repair deficient); patients with disease progression following checkpoint blockade; investment in AMV564 To achieve a steady-state exposure of 0.1 pM to 5 pM AMV564; the method involves administering 5 µg to 50 µg AMV564 by continuous intravenous infusion.

Methods for treating AML by administering AMV564 to achieve a steady state exposure of 30 pM to 70 pM AMV564 are also described.

CD33, also known as Siglec-3, is a transmembrane protein expressed on cells of the myeloid lineage. CD33 has long been considered an attractive target for acute myeloid leukemia (AML) due to its high prevalence and high expression on leukemic blasts. The function of CD33 is not well understood, but it has been shown that activation of CD33 signaling on early lineage myeloid cells, such as immunosuppressive myeloid-derived suppressor cells (MDSCs), leads to MDSC expansion and suppresses the production of cytokines and factors. CD33 expression was used as a component of a cell surface marker panel to identify these immunosuppressive monocytes and granulosa cells (eg, by immunophenotyping of the cells using flow cytometry). It is unclear what role, if any, CD33 plays on more differentiated myeloid lineage cells such as mature monocytes, neutrophils, macrophages and dendritic cells. Indeed, genetic knockout of CD33 in human cells using CRISPR technology has shown that CD33 is not required for lineage differentiation, yet it continues to be expressed in variable amounts in the majority of these cells, making it equally safe and efficacious. A challenging target for non-selective T cell engagers.

T cell adapters function by acting as a bridge between an antigen on the target cell and a different antigen on the T cell to form a ternary complex between the drug and the two different cell types, thus mimicking the process of adaptive immunity. Formation of natural T cell-target cell synapses generated during the reaction. It is believed that between 10 and 100 molecules of drug must be properly bound in order to activate T cell killing, and T cells must also be accessible. Thus, this activity state brings additional considerations for receptor occupancy and dosing beyond standard models, such as binding inhibitory ligands or receptors via biopharmaceuticals, where dosing strategies aim to achieve maximum target coverage until Unacceptable toxicity. For T cell engagers targeting broadly expressed antigens such as CD33, there are still more factors to consider. In addition to the potential safety risks associated with widespread depletion of cells of the myeloid lineage that confer valuable protection against infection, this population of cells can be very large (e.g., up to 100 billion neutrophils exist in the human body per day), and Efficacy may thus be compromised due to inadequate distribution of the drug and insufficient supply of T cells to accomplish the kill. Thus, targeting other cell populations such as leukemic blasts or even more rarely immunosuppressed MDSCs may thus be hampered by the broad distribution of the drug in CD33-positive normal myeloid cells and the need for sufficient relevant T cells to achieve kill. For these reasons, determining the appropriate dose, dosage regimen, and route of administration of T cell engagers is far more challenging than monovalent, monospecific agents.

Binding Properties and Dose Selection of AMV564 The bivalent design of AMV564 is reflected in its physical properties. AMV564 is extremely potent and exhibits cell killing at low receptor occupancy, ex vivo or in vitro with picomolar or sub-picomolar EC50 values for depletion of CD33 target cells . AMV564 is a potent agonist that induces biological activity at low receptor occupancy or low levels of target binding. Binding studies using flow cytometry showed the absence of neutrophils, multiple Binding of nucleated (PMN) leukocytes or monocytes (FIGS. 1F-1G). At concentrations of 1 pM and 10 pM, AMV564 appears to be highly selective for MDSCs compared to other abundant CD33 expressing cells.

Thus, when target cells engage AMV564 sufficiently for activity but with minimal binding to other myeloid cells, an ideal therapeutic window for safety and activity against tumor (e.g. leukemic blasts) and/or against suppressor (MDSC) cells can be achieved . In addition to safety considerations, over-engagement of large cell populations comprising normal myeloid lineages may lead to reduced efficacy due to suboptimal drug distribution and insufficient T cells available to form synapses that mimic native T cells necessary to facilitate cell killing Ternary complex. to kill

Depletion of MDSCs and dose selection to overcome the suppressive tumor microenvironment is a major challenge in immunotherapy. Key cellular effectors of the suppressive tumor microenvironment are MDSCs, which are associated with immune dysfunction, antitumor immunosuppression, and poor response to immunotherapy. MDSCs inhibit T cell and NK cell responses through various cytokines, active species and pathways. Furthermore, it inhibits efficient antigen presentation by dendritic cells in tumor-draining lymph nodes. In another aspect of the invention, AMV564 depletes MDSCs in the peripheral and bone marrow of AML patients at low doses. The rapid decline observed at low introduced doses of AMV546 indicates efficient binding and depletion of both monospheroid and spheroid MDSC populations. MDSCs are rare in the periphery of healthy adults and become markedly elevated in cancer patients. However, it remains a relatively rare cell compared to the general mature myeloid lineage, and its depletion and control efficacy is high when total receptor occupancy is unfavorable for the selectivity of bivalent T cell engagers such as AMV564 Doses may be lowered.

In patients with solid tumors, administration of AMV564 at doses of 5 µg to 50 µg produced near steady-state exposures ranging from 0.1 pM to 5 pM, effectively depleted MDSCs and promoted favorable CD4 and CD8 T cell activation profiles and cellular interferon environment to promote the restoration of anti-tumor immunity. Higher doses of 50 µg to 75 µg or 75 µg to 150 µg will also produce exposures that remain within the selective range for MDSC depletion.

Circulating MDSCs are pharmacodynamic biomarkers of AMV564 and T cell responses. Since MDSCs are known to be induced by T cell activation, they were induced due to T cell activation stimulated by AMV564. MDSC reflects engagement of AMV564 with target cells (MDSC) and depletion of such cells, and is dose-related to bivalent, bispecific T-cell engagers such as AMV564, which reflect effective doses within the drug's optimal therapeutic index , to enable efficient depletion of these relatively rare cells compared to the rest of the CD33-positive myeloid lineage.

Because AMV564 depletes MDSCs, treatment with AMV564, eg, according to the dosing regimens described herein, may be useful for depleting MDSCs in a variety of circumstances. For example, AMV564 can be used to deplete MDSCs in patients being treated with activated T cells or therapies involving the administration of activated T cells.

Although not fully understood, the management of cytokine release syndrome ( CRS ) appears to involve T cell activation and subsequent activation of macrophages and other myeloid cells to produce and secrete IL-6, IL-1B, and other cytokines . CRS is often associated with T cell engaging therapy, such as T cell engaging bispecific antibody and CAR-T therapy. CRS was most evident at the start of dosing. As shown below, administration of AMV564 by the subcutaneous route in patients with solid tumors, e.g., at doses of 15 μg to 50 μg, resulted in robust T cell activation as assessed by various measures, including detectable Measured peripheral interferon gamma (IFNγ) increased up to 10- to 40-fold relative to baseline. However, the increase in IL-6 was relatively small (approximately 1:1 of the two cytokines or in favor of higher IFNγ) (see Figures 7A-7E), and no significant levels of IL-1B were detected. This favorable profile is consistent with the absence of CRS observed in patients in this clinical study. This favorable pattern of apparently robust T cell activation and lack of CRS likely reflects a combination of features including depletion of MDSCs (which can produce inflammatory cytokines), engagement of bivalent T cells with AMV564 (which can more closely resemble more Naive T cell receptor engagement) and lymphatic delivery and distribution kinetics associated with subcutaneous injection of AMV564. AMV564 has a favorable therapeutic index suitable for long-term administration, and these properties should also help alleviate CRS after completion of lead-in dosing to the target dose.

Combination Therapy Effective treatment of neoplasia is often achieved through combination therapy. The functional therapeutic index of AMV564 at a dose range where efficacy and safety can be maximized (especially without significant depletion of normal myeloid cells) makes AMV564 suitable for combination therapy. In solid tumors, combination strategies include but are not limited to checkpoint blockade (PD-1 or PDL-1 blockade); T cell activators and expanders such as interleukins IL-2, IL-10, and IL -15; Dual targeting agents such as those targeting checkpoints (e.g. PD-1 or PDL-1) and immunosuppressors (e.g. TGFβ); CAR therapy (expressed in T cells or NK cells); NK activation therapy ; or standard-of-care chemotherapy. In AML and MDS, the previously listed therapies may also be used in combination with other established AML agents such as hypomethylating agents (e.g. azacytidine, decitabine), differentiating agents ( For example targeting IDH1/2), targeting agents (eg anti-FLT3), agents targeting anti-apoptotic proteins such as BCL2 (eg venetoclax), BCL-XL or MCL1 or lenalidomide (lenalidomide).

AMV564 can be used alone or in combination for the treatment of melanoma (e.g., patients with unresectable or metastatic melanoma, melanoma involving lymph nodes after complete resection); non-small cell lung cancer (NSCLC) (e.g., metastatic non- Squamous NSCLC, III NSCLC, metastatic NSCLC with PD-L1 expression); head and neck squamous cell carcinoma (HNSCC); classic Hodgkin lymphoma (cHL); primary mediastinal large B-cell lymphoma (PMBCL); urothelial carcinoma (e.g., locally advanced or metastatic urothelial carcinoma expressing PD-L1); high MSI carcinoma (e.g., unresectable or metastatic, high MSI- H) or mismatch repair deficiency; solid tumors that have progressed after prior therapy; breast cancer; uterine cancer; gastric cancer (eg, recurrent locally advanced or metastatic gastric or gastroesophageal junction adenocarcinoma expressing PD-L1); cervical cancer; liver Cell carcinoma (HCC); Merkel cell carcinoma (MCC); and renal cell carcinoma (RCC).

Cross References to Related Applications

This application claims the benefit of the following U.S. Provisional Application, filed April 9, 2021: Serial No. 63/173,224, which is hereby incorporated by reference in its entirety.

AMV564 AMV564 is a homodimer of SEQ ID NO:1. AMV564 is described in US 9212225 (diabody 16; SEQ ID NO: 113 without a 6 His tag at the amino terminus) and WO 2016/196230 (SEQ ID NO: 139). The pharmaceutical composition of AMV564 comprises a homodimer of a polypeptide having an amino acid sequence of SEQ ID NO: 1 and a pharmaceutically acceptable carrier or excipient.

Example 1 : AMV564 ex vivo depletes MDSCs and activates T cells In this study, AMV564 treatment of ex vivo primary cells (PBMCs, MDS bone marrow, tumor PBMCs) was found to deplete MDSCs and activate T cells. Figure 1A shows that treatment of PBMCs with the CD33 ligand S100A9 resulted in MDSC expansion and increased CD33 expression. Exposure to AMV564 counteracted reactive oxygen species (ROS) production in response to S100A9 stimulation of PBMCs to expand MDSCs (Fig. 1B), caused selective depletion of MDSCs (Fig. 1C), and increased CD8 T cells (Fig. 1D) and CD4 T cells. Cell (FIG. 1E) number and activation status (as assessed by IFNγ positive ratio).

Thus, ex vivo processing of patient-derived peripheral blood mononuclear cells (PBMCs) resulted in selective depletion of MDSCs (p < 0.01) and reduced production of reactive oxygen species. AMV564 induced a significant increase in activated T cells only in the presence of CD33+ target cells, with a >2-fold increase in CD4+ and CD8+ T cell proliferation. The increase in proliferation was dose-dependent and was accompanied by a marked increase in IFNy production.

AMV564 bound to MDSCs (and the leukemia blast cell line KG1) at 1 pM and 10 pM (Fig. 1F). At these concentrations, however, there was essentially no evidence of AMV564 binding to monocytes, neutrophils, and polymorphonuclear leukocytes (PMN). These concentrations were within the range of exposures observed when AMV564 was administered by the subcutaneous route at doses of 5-15-50 μg (approximately 0.1-5 pM). As shown in Figure 1G.

Example 2 : AMV564 Depletes MDSCs and Activates T Cells in Patients In this clinical study, it was found that treatment with AMV564 caused depletion of peripheral blood MDSCs and bone marrow MDSCs and AML blasts without reduction in neutrophils (Figure 2A-2F) . Rapid depletion of both monocytes and granulosa MDSCs was evident, with little or no effect on circulating neutrophil or monocyte populations. Evidence of early T cell activation was evident in the rapid redistribution/margination of T cells (this apparent transient lymphopenia was a consequence of T cell activation and migration to lymph nodes and tissues). In FIGS. 2A-2F , the period of introduction of AMV564 dosing (15 μg; 3 days) is represented by light bars and target AMV564 dosing (100 μg) is represented by dark bars. In MDSCs, depletion was observed in peripheral blood (Figure 2A and Figure 2B) and bone marrow (Figure 2C). Figures 2D to 2E show the effects of AMV564 treatment on peripheral blood T cells, peripheral blood neutrophils and peripheral blood blasts, respectively.

Example 3 : AMV564- depleted MDSCs in Solid Tumor Patients An initial increase in peripheral blood MDSCs in response to T cell activation was observed at the time of introduction of dosing (Day 1 to Day 3 in some patients) (Figure 3A-3C ). Rapid redistribution/bordering of peripheral blood T cells consistent with T cell activation was also observed as T cells migrated to lymph nodes and tissues (Fig. 3C). However, at the target dose, peripheral MDSCs were controlled. Bone marrow MDSCs were also substantially reduced relative to baseline when assessed at day 15. However, once AMV564 treatment was stopped, both bone marrow and peripheral blood MDSCs rebounded.

Example 4 : AMV564 is a selective and potent conditional agonist Primary human T cells and KG-1 cells were exposed to AMV564. Measure target-dependent cytotoxicity (Figure 4A), target-dependent T cell proliferation (Figure 4B), viability of differentiated monocytes and neutrophils (Figure 4C), viability of differentiated monocytes and neutrophils (FIG. 4D), all using CD3/CD28 as reference T cell stimulation. Since KG1 expresses CD33 and AMV564 binds KG1 similarly to MDSC, KG1 was used as a surrogate for MDSC in these assays. AMV564 induced potent dose-dependent cell death of KG1 at maximal levels similar to CD3-CD28 stimulation (Fig. 4A). This was accompanied by an increase in daughter cells, reflecting levels of T cell proliferation equal to or exceeding the CD3-CD28 reference stimulus (Fig. 4B). However, unlike general T cell stimulation using CD3-CD28, there is no evidence that AMV564 promotes significant cell death of autologous monocytes or neutrophils (Figure 4C), and similarly, there is no evidence that these cells Population induced T cell proliferation (Fig. 4D).

Example 5 : Phase 1 Clinical Study of AMV564 in Patients with Solid Tumors This study enrolled adult patients with unresectable advanced metastatic solid tumors that had recurred and progressed since last antineoplastic therapy without recognized standard therapy. Patients had an ECOG performance status of ≤2 with adequate organ function. Patients were treated with AMV564 (15, 50 or 75 μg/day) alone or with AMV564 (5, 15 or 50 μg/day) in combination with pellizumab administered intravenously at 200 mg every 3 weeks (Q3W) treat. In both cases, AMV564 was administered once a day by subcutaneous injection on days 1 to 5 and days 8 to 12 of a 21 day cycle. AMV564 was well tolerated and pharmacodynamic analyzes showed evidence of relief of immunosuppression (reduction of MDSC and Treg) and promotion of effector CD8 and Th1 CD4 responses in a phase 1 population of heterogeneous cancer patients previously treated with other therapies.

In general, patients treated with AMV564 exhibited a cytokine profile consistent with the activation of T cells including CD4 Th1 helper cells, antigen-presenting cells, and improved migration of T cells toward tissues such as tumor tissues (IFNγ, IL -15, IL-18, soluble granzyme B and CXCL10 increase). Although strong T cell activation was observed, no interleukin release syndrome episodes occurred.

Example 6 : MDSC control and Treg control in patients with solid tumors related to M-MDSC and G-MDSC in patients with solid tumors and with AMV564 (Fig. : small intestine-15 μg AMV564; Figure 5D: gastroesophageal junction-15 μg AMV564) was controlled in patients treated with AMV564 (on days 1 to 5 and days 8 to 12 of a 21-day cycle subcutaneous injection once a day). Figure 5E depicts the change in Treg from baseline (B) over two therapy cycles (C1 and C2).

Example 7 : Increased CD8 to Treg Ratio in Solid Tumor Patients Receiving AMV564 Therapy Figures 6A to 6G show that for the majority of solid tumor patients receiving AMV564 therapy, an increased CD8/Treg ratio was observed (during days 1 through 21 of the 21-day cycle). Subcutaneous injection once a day on day 5 and days 8 to 12 (Fig. 6A: small intestine - stable disease (15 μg dose); Fig. 6B: ovary - complete response (15 μg dose); Fig. 6C: GE junction - disease progression (15 μg dose); Figure 6D: Endometrium - stable disease (50 μg dose); Figure 6E: Colorectum - progressive disease (50 μg dose); Figure 6F: Skin - stable disease (50 μg dose); Figure 6G : appendix-stable disease (50 μg dose)).

Example 8 : AMV564 Promotes Favorable CD4 and CD8 T Cell Polarization in Ovarian Cancer Patients Previously Received Multiple Platinum-Based Chemotherapy, Surgery, Radiation, Pembrolizumab (Best Response Stable Disease, 6 Months Before Study Start Months completed) and niraparib and letrozole regimens in ovarian cancer patients received 15 µg of AMV564 (on days 1 to 5 and days 8 to 12 of a 21-day cycle by subcutaneous injection once a day). This patient showed a sustained increase in CD8/Treg, increased % CD8, unchanged or increased effector CD8 (TBX21 and/or granzyme B positive) and memory CD8 cells, a dynamic increase in TBX21-positive CD4 T helper cells, and a proportion of PD1-positive CD8 Dynamic modulation, but no substantial increase overall, as shown in Figures 7A-7E. This patient progressed from stable disease to partial response to complete response as assessed by CT scans performed at 6-8 week intervals.

Example 9 : Patients treated with AMV564 showed signs of T cell activation without CRS Figures 8A to 8F (patients 1, 2, 3, 11 , 9 and 14, respectively) show IFNγ and IL-6 measurements in six patients with solid tumors treated by subcutaneous injection once a day to day 5 and days 8 to 12. As can be seen, there is clear evidence of systemic IFNy production without excess IL-6 production (IFNy:IL-6 ratio of about 1:1 or better for most patients).

During the 5 treatment cycles, patients with solid tumors (data not shown) showed increases in IFNγ, TNFα, and IL18 and other factors at later time points (such as the end of cycle 2 and cycles 3 and 4), compared with I MHC-like upregulation, dendritic cell activation, and T-cell migration (decreased MDSC-inducing factor G-CSF) were consistent. This same patient entered the study with suboptimal CD8/Treg at baseline. Improvements were observed throughout the course of treatment, especially around cycle 3, where an increase in CD8 T cell proliferation and activation (Ki67 and CD38 ratio) was observed in relation to CD8 effector function (T-bet and granzyme B positive ratio) Improve consistency. The timing of this observed increase in cytokines and factors is consistent with improved dendritic cell activation and Th1 responses, suggesting that AMV564 drives more favorable immune polarization over time even in this advanced patient.

Example 10 : Treatment with AMV564 and Pilletizumab Figures 9A to 9D show M-MDSC and G-MDSC measurements in four solid tumor patients treated with AMV564 in combination with Pilletizumab, AMV564 at 21 The 1st to 5th day and the 8th to 12th day of the daily cycle were subcutaneously injected once a day (5 µg/day (Figure 9A and Figure 9B) or 15 µg/day (Figure 9C and Figure 9D). Antibiotics were administered intravenously at 200 mg every 3 weeks (Q3W). AMV564 administration days are indicated by bars along the x-axis and pembrolizumab treatment days are indicated by asterisks. As can be seen, excellent MDSC control was observed.

Figures 10A to 10D show the results of treatment with AMV 564 (15 µg subcutaneously once daily on days 1 to 5 and 8 to 12 of a 21-day cycle) and pembrolizumab (200 mg Q3W intravenously). Administered) the data of two patients (Figure 10A and Figure 10C: patient 15; Figure 10B and Figure 10D: patient 16) of combination treatment. This data shows evidence of a substantial increase in the proportion of CD8 effector cells and a substantial increase in the ratio of CD8/Treg in cycles 1-2. The data also showed expansion of T-Bet and granzyme B positive CD8 cells. These effects were not evident in the 5 μg AMV564 combination cohort in this study.

Figure 11A and Figure 11B show the effect of AMV 564 (15 µg subcutaneously once daily on days 1 to 5 and days 8 to 12 of a 21-day cycle) with pembrolizumab (200 mg Q3W intravenously). CD8 T cell proliferation data of two patients who were administered the combination therapy ( FIG. 11A : patient 15; FIG. 11B : patient 16). This data shows evidence for a significant increase in CD8 proliferation (assessed by CD8 Ki67) and activation (assessed by CD8CD38). 2 of 3 combination-dosed patients showed significant and rapid increases from unfavorable baseline levels, suggesting a potential benefit of the combination of AMV564 and pembrolizumab.

Example 11 : AMV564 Selectively Targets M - MDSCs and G - MDSCs to Deplete and Activate T Cells in Solid Tumor Patients -MDSC. As can be seen in Figures 12A-12B, treatment was associated with a decline in both MDSC subtypes. This is important because elevated M-MDSCs are often associated with lower levels of peripheral T cells.

Treatment of patients with solid tumors with AMV564 (alone or in combination with pembrolizumab) increased the co-expression of granzyme B and TBX21 (T-bet) on CD8+ T cells ( FIG. 13A ). Furthermore, the frequency of granzyme B+ CD8+ T cells increased significantly between the first and second cycle in treated patients (Fig. 13B)

Example 12 : AMV564 induces modulation of immune responses Alone or in combination with pializumab Solid tumor patients treated with 15 μg or 50 μg of AMV564 (n=11 monotherapy, n=4 combination) showed favorable, approximately 1:1 IFNy:IL-6 ratio (Figure 14A and Figure 14B). In many cases, treatment with other T cell engagers yielded ratios between 0.1 and 0.01.

In patients with solid tumors treated with AMV564, the levels of IL-6, IL-1β, IL-10 and TNFα (all of which are myeloid-derived cytokines) remained low (Fig. 15A, Fig. 15B and data not shown ). In contrast, in these patients, levels of pro-inflammatory cytokines that promote Th1 polarization, macrophage activation, and T cell migration to tumors were elevated (Fig. 15A, Fig. 15B and data not shown).

In vitro cytotoxicity assays using KG-1 cells as target cells showed that AMV564 was associated with a favorable IFNy:IL-6 ratio over a wide range of AMV564 ratios (Fig. 15C).

Example 13 : AMV564 Expansion of Peripheral T Cell Repertoire via TCRβ CDR3 Deep Sequencing in Three Patients (Small Intestinal Cancer, Squamous Cell cancer and pancreatic cancer) T cell repertoire was assessed. The clonality of expansion, restriction or de novo generation during treatment was assessed to correlate the effect of treatment on TCR repertoire and disease evolution. As can be seen in Figures 16A-16C, a significant expansion of the T cell repertoire was evident after only one treatment cycle (p=0.008). Each patient was observed to have approximately 30 to over 300 differentially detected T-cell clones, including some that were undetectable or very rare at baseline.

Example 14 : T cell repertoire expansion in ovarian cancer patients is associated with increased CD8 memory cells. A confirmed RECIST CR ovarian cancer patient treated with 15 µg AMV564 showed an increase in CD8 cells (Fig. 17A) and CD8 memory cells (Fig. 17B) during treatment.

Tracking of specific T cell rearrangements across treatment time points indicated that several clones expanded and eventually dominated this patient's cell pool (Figure 17C). Two of the eight most expanded T cell clones matched CDR3 sequences consistent with T cells targeting the SLC3A2 neoantigen (upregulated in certain cancers and often associated with poor prognosis) (Fig. 17C).

INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. way incorporated. In case of conflict, the present application, including any definitions herein, will control.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the claims below.

Figures 1A - 1G present data showing that AMV264 depletes MDSCs and activates T cells ex vivo, and that AMD564 binds MSCs and KG-1 cells at 1 pm and 10 pm, but not monocytes at these concentrations. Figure 1A shows that treatment of PBMCs with the CD33 ligand S100A9 resulted in MDSC expansion and increased CD33 expression. Figures 1B-1E show that exposure to AMV564 counteracts the production of reactive oxygen species (ROS) in response to S100A9 stimulation of PBMCs to expand MDSCs (Figure 1B), causes selective depletion of MDSCs (Figure 1C), and increases CD8 T Cell (Fig. 1D) and CD4 T cell (Fig. 1E) numbers and activation status (as assessed by IFNγ positive ratio). Figures 2A - 2F show that AMV564 treatment resulted in the depletion of peripheral blood and bone marrow MDSCs and AML blasts without neutrophil reduction. Figures 2A and 2B show depletion of MDSCs in peripheral blood. Figure 2C shows MDSC depletion in bone marrow. Figures 2E to 2G show the effect of AMV564 treatment on peripheral blood T cells (Figure 2E), peripheral blood neutrophils Figure 2F and peripheral blood blasts (Figure 2G). Light colored bars indicate the days on which the dose was introduced, and dark bars indicate the days on which the target dose was introduced. Figures 3A to 3C present data showing the effects of AMV564 on peripheral blood MDSCs, bone marrow MDSCs, and peripheral blood T cells. Figure 3A shows the effect of AM564 on the percentage of CD45+ cells in peripheral blood MDSCs. Figure 3B shows the effect of AM564 on the percentage of CD45+ cells in bone marrow MDSCs. Figure 3C shows the effect of AM564 on the percentage of CD45+ cells in peripheral blood T cells. Light colored bars indicate the days on which the dose was introduced, and dark bars indicate the days on which the target dose was introduced. The data presented in Figures 4A - 4D demonstrate that AMV564 is a selective and potent conditional agonist. Single open circles and triangles show the results of CD3/CD28 stimulation in the absence of AMV564. Figure 4A shows that AMV564 induced potent dose-dependent cell death of KG1 at a maximal level similar to CD3-CD28 stimulation (Figure 4A). Figure 4B shows an increase in daughter cells, reflecting levels of T cell proliferation equal to or exceeding the CD3-CD28 reference stimulus. Figure 4C shows that there is no evidence that AMV564 promotes significant cell death in autologous monocytes or neutrophils. Figure 4D shows that, unlike general T cell stimulation using CD3-CD28, there is no evidence that these cell populations induce T cell proliferation. Figures 5A - 5E present data showing that MDSC control correlates with Treg control in solid tumor patients. Figure 5A to Figure 5D show that M-MDSC and G-MDSC were treated with AMV564 in solid tumors (Figure 5A: ovary-15 μg AMV564; Figure 5B: skin-50 μg AMV564; Figure 5C: small intestine-15 μg AMV564; Figure 5D: Gastroesophageal junction - 15 μg AMV564) treated patients were controlled with AMV564 (once daily subcutaneous injections on days 1 to 5 and days 8 to 12 of a 21 day cycle). Solid squares are G-MDSC and solid circles are M-MDSC. Figure 5E depicts the change in Treg from baseline (B) over two therapy cycles (C1 and C2). The bars on the axis indicate the days of administration of AMV564. Figures 6A - 6G present data showing that the CD8:Treg ratio was improved by AMV564 therapy in patients with solid tumors. Specifically, Figures 6A to 6G show that increased CD8/Treg ratios were observed for the majority of solid tumor patients receiving AMV564 therapy (once a day on days 1 to 5 and days 8 to 12 of a 21-day cycle). Subcutaneous injection (Fig. 6A: small bowel - stable disease (15 μg dose); Fig. 6B: ovary - complete response (15 µg dose); Fig. 6C: GE junction - progressive disease (15 µg dose); Fig. 6D: endometrium - stable disease (50 μg dose); Figure 6E: colorectum - progressive disease (50 μg dose); Figure 6F: skin - progressive disease (50 μg dose); Figure 6G: appendix - stable disease (50 μg dose)). Dotted lines represent baseline ratios; bars along the x-axis represent days of dosing and wider bars represent ratios of healthy controls whose peripheral blood samples were processed in the same flow-based assay. Figures 7A - 7E present data showing AMV564 in Administration of 15 μg AMV564 (injected subcutaneously once daily on days 1 to 5 and days 8 to 12 of a 21 day cycle) promoted favorable CD4 and CD8 T cell polarization. Figure 7A shows 150 CD8/Treg ratio within days. Figure 7B shows dynamic modulation of effector CD8 (TBX21 and/or granzyme B positive) and PD1 positive CD8 ratios maintained or increased. Figure 7C shows dynamic increase in TBX21 positive CD4 T helper cells Figure 7D shows a sustained increase in T cells. Figure 7E shows an increase in the percentage of CD8 T cells. The dotted line represents the baseline ratio; the bars along the x-axis represent the days of administration and the wide bars represent the ratio of healthy controls. Fig . 8A to Fig. 8F show IFNγ Cycle 1, IFNγ Cycle 2, IL-6 Cycle 1, and IL-6 Cycle 2 Levels in Six Solid Tumor Patients Treated with AMV564 (Patient 1 (Fig. 8A); Patient 2 (Fig. 8B) ; Patient 3 (Figure 8C); Patient 11 (Figure 8D); Patient 9 (8E); Patient 14 (Figure 8F - Cycle 1 only)). Figures 9A to 9D show the results of four patients treated with AMV564 and palizumab Anti-(pembrolizumab) combination therapy M-MDSC and G-MDSC measurement results in solid tumor patients. Figure 9A and Figure 9B show the results observed in solid tumor patients treated with AMV564 and pembrolizumab combination, AMV564 was injected subcutaneously once a day at 5 μg/day on days 1 to 5 and days 8 to 12 of a 21-day cycle, respectively, and pilizumab was administered intravenously at 200 mg every 3 weeks (Q3W) Figure 9C and Figure 9D show the results observed for patients with solid tumors treated with AMV564 in combination with pembrolizumab, AMV564 on days 1 to 5 and days 8 to 12 of a 21-day cycle at 15 μg/day was injected subcutaneously once a day, and pilizumab was administered intravenously at 200 mg every 3 weeks (Q3W). Heart squares are G-MDSCs, and solid circles are M-MDSCs. The bars on the axis indicate the days of AMV564 administration. Figures 10A - 10D show the effect of the combination of AMV564 and pembrolizumab on T-Bet and granzyme B positive CD8 cells and CD8/Treg ratios in two solid tumor patients. Figure 10A and Figure 10C show the contrast AMV 564 (subcutaneous injection of 15 µg once a day on days 1 to 5 and days 8 to 12 of a 21-day cycle, respectively) and pembrolizumab (200 mg Q3W via Intravenous administration) observed results for patient 15 of combination therapy. Figure 10B and Figure 10D show the comparison between AMV 564 (15 µg subcutaneously injected once a day on days 1 to 5 and days 8 to 12 of a 21-day cycle) and pembrolizumab (200 mg Q3W intravenously). The results observed for patient 16 treated with the combination therapy. Dashed lines represent baseline rates; bars along the x-axis represent days of AMV564 dosing and broad bars represent rates of healthy controls. Figures 11A - 11B show the effect of combination therapy of AMV564 and pembrolizumab on CD8 cell proliferation and activation in two solid tumor patients. Figure 11A and Figure 11B show the contrast between AMV 564 (15 µg subcutaneously injected once a day on days 1 to 5 and days 8 to 12 of a 21-day cycle) and pembrolizumab (200 mg Q3W intravenously). The results observed for patient 15 (FIG. 11A) and patient 16 (FIG. 11B) administered the combination treatment. 12A to 12B show the effect of AMV564 on the content of M-MDSC cells and G - MDSC cells during 5 treatment cycles (* p < 0.05, **p < 0.01). Figure 12A shows the effect of treatment of solid tumor patients with AMV564 administered subcutaneously at 15 μg or 50 μg on M-MDSC levels. Figure 12B shows the effect on G-MDSC content of solid tumor patients treated with 15 μg or 50 μg subcutaneously administered AMV564. Figures 13A - 13B show the effect of AMV564 alone or in combination with pellizumab on co-expression of granzyme B and TBX21 on CD8+ T cells (Figure 13A) and frequency of granzyme B+ CD8+ cells (Figure 13B). Figures 14A - 14B show the effect of AMV564 alone or in combination with pembrolizumab on IFNγ and IL-6 levels. Figure 14A shows the levels of IFNγ and IL-6 observed in patients with solid tumors treated with 15 μg or 50 μg of AMV564 alone or in combination with pembrolizumab (n=11 monotherapy, n=4 combinations) . Figure 14B shows that AMV564 exhibits a favorable IFNγ:IL-6 ratio of approximately 1:1 relative to other T cell engagers. Figures 15A - 15C show the effect of AMV564 alone or in combination with pembrolizumab on the levels of various cytokines in patients and in in vitro cytotoxicity assays. Figure 15A shows the effect of AMV564 alone or in combination with pembrolizumab on TNFα, IL-1β and IL-10 levels in patients. Figure 15B shows the effect of AMV564 alone or in combination with pembrolizumab on the levels of IP-10 (CXCL10). Figure 15C shows the results of the AMV564 cytotoxicity assay performed using KG-1 cells as target cells. Figures 16A - 16C show the effect of AMV564 alone or in combination with pembrolizumab on the T cell repertoire of three different patients. Figure 16A shows the expansion of the T cell repertoire in small bowel cancer patients. Figure 16B shows the expansion of the T cell repertoire in patients with squamous cell carcinoma of the penis. Figure 16C shows the expansion of the T cell repertoire in pancreatic cancer patients. Orange circles indicate inbred lines that were significantly amplified or not detectable at baseline. Figures 17A to 17C show the effect of AMV564 on rearrangement of CD8 and CD8 memory cells and T cells in ovarian cancer patients confirmed as RECIST CR. Figure 17A shows the increase in CD8 cells during treatment in ovarian cancer patients treated with 15 μg AMV564. Figure 17B shows the increase in CD8 memory cells during treatment in ovarian cancer patients treated with 15 μg AMV564. Figure 17C shows tracking of specific T cell rearrangements across treatment time points in ovarian cancer patients.

Claims (41)

A method of treating a patient suffering from a solid tumor, the method comprising administering to the patient immunotherapy and AMV564, wherein AMV564 is administered before, after, or together with the immunotherapy. The method according to claim 1, wherein AMV564 is administered by subcutaneous injection. The method of claim 1, wherein the AMV564 is administered within 4 to 6 weeks after the administration of the immunotherapy. The method according to claim 3, wherein the AMV564 is administered for at least 7 days within 14 days. The method according to claim 4, wherein the AMV564 is administered for 10 days within 14 days. The method according to claim 5, wherein the AMV564 is continuously administered in two periods within 14 days for 5 days. The method according to claim 5, wherein the AMV564 is continuously administered for 5 days, followed by no administration for the next two days, and then continuously administered for 5 days. The method of any one of claims 4 to 7, wherein the AMV564 is administered within a 21-day cycle, wherein AMV564 is administered for at least 7 days within a 14-day period and not administered within the subsequent 7-day period. The method of claim 8, wherein the 21-day cycle is repeated at least twice. The method according to claim 9, wherein AMV564 is administered for at least 10 days within 14 days, wherein the administration is continued for 5 days, followed by non-administration for 2 days, and then continuous administration for 5 days. The method according to any one of claims 1 to 10, wherein when administering, the dose of AMV564 administered per day is 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 μg. The method of claim 1, wherein AMV564 is administered at 15 μg/day for 5 days within the first week of therapy, and then 50 μg is administered once a week thereafter. The method as claimed in item 1, wherein 5 μg to 50 μg of AMV564 is administered once a week. The method of claim 1, wherein AMV564 is administered at 15 µg/day for 5 days within the first week of therapy, and then 15 µg is administered once a week thereafter. The method of claim 1, wherein AMV564 is administered at 15 μg/day for 5 days within the first week of therapy, and then 15 μg to 50 μg is administered once a week thereafter. The method according to any one of claims 1 to 15, wherein the immunotherapy is CR T cell therapy, CTL therapy and antibody therapy. The method according to claim 1, wherein the solid tumor is selected from breast cancer, pancreatic cancer, ovarian cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, urothelial cancer, squamous cell carcinoma, rectal cancer, penile cancer, uterus Endometrial cancer, small bowel cancer, appendix cancer. The method of claim 1, wherein the administration of AMV564 achieves a steady state exposure of 0.1 pM to 5 pM AMV564. The method of claim 1, wherein the administration of AMV564 achieves a steady state exposure of 0.5 pM to 3 pM AMV564. The method of claim 1, wherein the administration of AMV564 achieves a steady state exposure of 1 pM to 5 pM AMV564. The method according to claim 1, wherein the immunotherapy is anti-PD-L1 antibody or anti-PD-1 antibody. The method according to claim 21, wherein the anti-PD-1 antibody is nivolumab, pembrolizumab and cemiplimab. The method according to claim 21, wherein the anti-PD-L1 antibody is atezolizumab, avelumab or durvalumab. The method of claim 1, wherein the immunotherapy is CAR T cell therapy and AMV564 is administered 1 to 5 days, 5 to 10 days, or 5 to 14 days after administration of the CAR T cell therapy. A method of treating cancer in a patient, the method comprising administering AMV564 to the patient, wherein the cancer does not express CD33. The method of claim 25, wherein AMV564 is administered by subcutaneous injection. The method of claim 25 or 26, wherein AMV564 is administered at 15 μg/day for 5 days within the first week of therapy, and then 50 μg is administered once a week thereafter. The method as claimed in item 25, wherein 50 µg of AMV564 is administered once a week. The method of claim 25, wherein AMV564 is administered at 15 µg/day for 5 days within the first week of therapy, and then 15 µg is administered once a week thereafter. The method of claim 25, wherein AMV564 is administered at 15 µg/day for 5 days within the first week of therapy, and then administered at 15 µg to 50 µg once a week thereafter. The method according to claim 25, wherein the dose of injected AMV564 is 5 μg to 50 μg. The method according to any one of claims 25 to 31, wherein the solid tumor is selected from pancreatic cancer, ovarian cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, urothelial carcinoma, squamous cell carcinoma, rectal cancer, Cancer of the penis, endometrium, small intestine and appendix. The method according to any one of claims 25 to 31, wherein the solid tumor is selected from small cell lung cancer (NSCLC) (such as metastatic non-squamous NSCLC, III NSCLC, metastatic NSCLC expressing PD-L1), melanoma , Merkel cell carcinoma, MSI-high cancer (e.g., unresectable or metastatic, MSI-H, or mismatch repair deficient); post-checkpoint blockade Progressive patients. The method according to any one of claims 1 to 31, wherein the patient has a cancer selected from the group consisting of: melanoma (for example, a patient with unresectable or metastatic melanoma, melanoma involving lymph nodes after complete resection) ; non-small cell lung cancer (NSCLC) (eg, metastatic nonsquamous NSCLC, III NSCLC, metastatic NSCLC expressing PD-L1); head and neck squamous cell carcinoma (HNSCC); classical Hodgkin's lymphoma (Classical Hodgkin's lymphoma) Lymphoma) (cHL); primary mediastinal large B-cell lymphoma (PMBCL); urothelial carcinoma (eg, locally advanced or metastatic urothelial carcinoma expressing PD-L1); high MSI carcinoma (eg, unresectable Sexual or metastatic, microstellar instability-high (MSI-H) or mismatch repair deficient; solid tumors that have progressed after prior therapy; gastric cancer (eg, recurrent locally advanced or metastatic gastric or gastroesophageal junction tumors expressing PD-L1 adenocarcinoma); cervical cancer; hepatocellular carcinoma (HCC); Merkel cell carcinoma (MCC); and renal cell carcinoma (RCC). A method for expanding T cells, the method comprising culturing the T cells in the presence of AMV564. The method of claim 35, wherein the T cells express chimeric antigen receptors. The method according to claim 35 or 36, wherein the culturing lasts for at least 5 days. A method for expanding NK cells, comprising culturing the NK cells in the presence of AMV564. The method of claim 38, wherein the NK cells express chimeric antigen receptors. The method according to claim 38 or 39, wherein the culturing lasts for at least 5 days. A method of expanding cytotoxic lymphocytes (CTL), the method comprising culturing the CTL in the presence of AMV 564.

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