JP2024513915A - Method of administering bispecific T cell engagers - Google Patents

Abstract

Methods for reducing myeloid-derived suppressor cells and activating T cells in a patient and for treating a patient with a solid tumor are described. These methods involve administering a CD3/CD33 T cell engager.

Description

Cross-Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63/173,224, filed April 9, 2021, the entire contents of which are incorporated herein by reference. be done.

Background T cell engagers, a specific class of bispecific antibodies, mediate binding between target cells and T cells, resulting in lysis by the T cell, as well as T cell activation, differentiation, and proliferation. . Although T-cell engagers exhibit significant efficacy and antitumor activity in some settings, a barrier to widespread therapeutic success is often their undesired activity against normal cells expressing the target of interest. It is. This "on-target, off-tumor" toxicity can be significant and has been widely reported with engineered T cell engagers.

Myeloid-derived suppressor cells (MDSCs) act locally and systemically to inhibit antitumor immunity, inhibiting effector T cell responses, promoting the formation of immunosuppressive regulatory T cells, and promoting dendritic Suppresses cell maturation and antigen presentation and promotes metastasis formation. MDSCs induce a variety of suppressive functions that inhibit normal T cell responses and cause refractoriness to immune checkpoint blockade. The primary function of MDSCs is to suppress T cell activity in various ways depending on the pathology and context. The presence of MDSCs is believed to be associated with poor outcome and lack of response to certain therapies, such as those that activate T cells and those associated with the use of checkpoint inhibitors.

Some T cell activation therapies (eg, immunotherapies such as T cell engager and CAR T cell therapies) are associated with cytokine release syndrome (CRS). The occurrence of CRS can limit the usefulness of some immunotherapies. Activation of T cells promotes activation of myeloid cells and production of various cytokines and chemokines, including IL-6. In some cases, cytokine and chemokine levels are of pathological grade. MDSCs belong to myeloid cells, which are the main producers of IL-6.

SUMMARY Described herein are methods for using AMV564, a bispecific, bivalent molecule that binds CD3 and CD33. AMV564 is a homodimeric protein (i.e., a polypeptide having the amino acid sequence of SEQ ID NO: 1) that has four single chain variable fragment (scFv) binding sites, two that bind CD33 and two that bind CD3. homodimer). Bivalent designs could theoretically restore selectivity to T cell engagers found in regions of high local target density, e.g. sites of active signaling or associated with high receptor density or expression. regions can be directed to bind preferentially. Although AMV564 binds to CD33, which is widely expressed in the myeloid lineage, selective binding of MDSCs can be used to administer AMV564 to provide a desirable therapeutic index. Importantly, AMV564 was found to be selective over a wide dose range. Without being bound to any particular theory, this may be due to some combination of bivalency, scFv affinity, and homodimer geometry. For example, and again without being bound to any particular theory, the structure of AMV564 allows it to bind to clusters of dimerized CD33.

AMV564 has dual activity, inducing T cell-mediated killing of MDSCs and promoting T cell activation, thereby promoting better polarization (e.g., Th1 CD4 T cells and effector CD8 T cells). . AMV564 has an EC50 against MDSCs of less than about 3 pM. When properly administered, AMV564 leads to the killing of MDSCs, with sufficient sparing of neutrophils, monocytes, and many differentiated myeloid cells, thereby inhibiting MDSC-mediated suppressive pathways.

AMV564 is useful in reducing MDSCs and can be used to reduce systemic immunosuppression, eg, in solid tumor patients. AMV564 can also be used in combination with various immunotherapies, both to control CRS and to reduce immunosuppression.

Peripheral MDSCs may play an important role in suppressing T cells and transporting T cells to tumor sites, which is a potential rate-limiting factor for therapies based on T cell activation. For example, in some situations, a 15 μg dose of AMV564 can significantly deplete the MDSC population. Because MDSCs are replenished from the bone marrow, peripheral depletion allows sufficient control to benefit antitumor immunity over a period of administration that exceeds the limited life span of tissue-resident MDSCs. However, distribution of AMV564 in the tumor microenvironment allowed targeting of MDSCs at the tumor site while promoting local proliferation of T cells. Furthermore, for example, doses of up to 50, 75, and 100 μg of AMV564 delivered or infiltrated by CIV route to draining lymph nodes in addition to the periphery allowed disinhibition of antitumor T cells and restoration of antigen presentation and immune homeostasis. For example, subcutaneous administration of AMV564 at doses of 5, 15, or 50 μg provides a direct mechanism for initial distribution in the lymphatic system, including tumor-draining lymph nodes. Lower doses of AMV564 are effective when administered subcutaneously, presumably due to access to the lymphatic system.

AMV564 can reduce immunosuppression and activate T cell effector function in cancer patients. AMV564 may do so by alleviating immunosuppression through targeted depletion of myeloid-derived suppressor cells (MDSCs) and directly activating/repolarizing T cells to improve T effector function. .

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

Described herein is a method for reducing myeloid-derived suppressor cells and activating T cells in a patient (e.g., a patient undergoing immunotherapy) comprising: peptide) to the patient. In various embodiments, AMV564 is administered by subcutaneous injection; the dose of AMV564 injected is 5-150 μg (micrograms); , 10, 11, 12, 13, or 14 days); AMV6564 was administered subcutaneously for 10 days during a 14-day period; AMV6564 was administered twice for 5 consecutive days during a 14-day period; AMV6564 was administered for 5 consecutive days, followed by a 2-day administration. AMV564 is administered in a 21-day cycle, and within the 21-day cycle, AMV564 is administered for at least 7 days during a 14-day period, and then is not administered for 7 days; The cycle is repeated at least twice; AMV564 is administered for at least 10 days during a 14-day period, with 5 consecutive days administered, then 2 days not administered, then 5 consecutive days administered; The doses of AMV564 administered are 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μg for each day administered, and 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 is a T cell engager that contains a CD3 binding domain and activates T cells. the patient has or is undergoing treatment for leukemia (acute myeloid leukemia or myelodysplastic syndrome); the patient has a solid tumor; or Solid tumors include pancreatic cancer, ovarian cancer, colon cancer, rectal cancer, non-small cell lung cancer, urothelial cancer, squamous cell cancer, rectal cancer, penile cancer, selected from the group consisting of endometrial cancer, small intestine cancer, and appendiceal cancer; by administration of the AMV564, 0.1 to 5 pM (for example, 0.1, 0.2, 0.3, 0.4, A constant exposure of AMV564 of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 pm) is achieved. .

Also described is a method for treating a solid tumor in a patient, the method comprising administering to the patient AMV564 (a polypeptide having the amino acid sequence of SEQ ID NO: 1). In various embodiments, AMV564 is administered by subcutaneous injection; the dose of AMV564 injected is 5-150 μg (mcg or micrograms); 9, 10, 11, 12, 13, or 14 days); AMV6564 was administered subcutaneously for 10 days during a 14-day period; AMV6564 was administered twice for 5 consecutive days during a 14-day period; AMV6564 was administered for 5 consecutive days, then 2 AMV564 is administered in a 21-day cycle, and is administered for at least 7 days during a 14-day period of the 21-day cycle, and then is not administered for 7 days; The 21 day cycle is repeated at least twice; AMV564 is administered for at least 10 days during a 14 day period, with 5 consecutive days administered, then 2 days not administered, then 5 consecutive days administered. the doses of AMV564 administered are 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μg for each day administered, and the patient is 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 is a T cell enzyme that contains a CD3 binding domain and activates T cells. the patient has or is being treated for leukemia (acute myeloid leukemia or myelodysplastic syndrome); the patient has a solid tumor; solid tumors include pancreatic cancer, ovarian cancer, colon cancer, rectal cancer, non-small cell lung cancer, urothelial cancer, squamous cell cancer, rectal cancer, and penile cancer. administration of AMV564 achieved a steady-state exposure of AMV564 of 0.1-5 pM; ) (e.g., metastatic non-squamous NSCLC, stage III NSCLC, metastatic NSCLC expressing PD-L1), melanoma, Merkel cell, frequent microsatellite instability cancers (e.g., unresectable or metastatic, microsatellite instability-high (MSI-H) or mismatch repair deficiency); patients are undergoing checkpoint blockade and administration of AMV564 results in steady-state exposure to 0.1-5 pM AMV564. achieved; the method involves administering 5-50 μg of AMV564 by continuous intravenous infusion.

Also described are methods for treating AML by administering AMV564 to achieve a steady-state exposure of 30-70 pM AMV564.

CD33, also known as Siglec-3, is a transmembrane protein and is expressed on cells of the myeloid lineage. CD33 has long been viewed as an attractive target in acute myeloid leukemia (AML) due to both its high expression and prevalence on leukemic blasts. Although the function of CD33 is not fully understood, it activates CD33 signaling on early lineage myeloid cells, such as immunosuppressive myeloid-derived suppressor cells (MDSCs), which promotes the proliferation of MDSCs as well as suppressive cytokines and suppression. It has been shown to result in the production of sex factors. Expression of CD33 is one of a set of cell surface markers for identifying these immunosuppressive monocytic and granulocytic cells (e.g., by using flow cytometry for cellular immunophenotyping). Used as an ingredient. 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, knockout of CD33 in human cells using CRISPR technology shows that although CD33 is not required for lineage differentiation, it continues to be expressed at varying levels on most of these cells, resulting in safety and efficacy. In both respects, it has been shown to be a difficult target for non-selective T cell engagers.

T cell engagers function by acting as a bridge between an antigen on a target cell and a different antigen on a T cell, forming a ternary complex between the drug and two different cell types, thereby mimicking the formation of a natural T cell-target cell synapse that occurs during the adaptive immune response. It has been suggested that 10-100 molecules of drug must be appropriately bound and available to the T cell to activate killing by the T cell. This active state therefore brings additional considerations for receptor occupancy and dosing beyond the standard model of, for example, inhibiting a ligand or receptor via biological drug binding, where the dosing strategy aims for maximum target coverage before reaching unacceptable toxicity. There are even more factors to consider for T cell engagers that target a broadly expressed antigen such as CD33. Apart from the potential safety risks associated with widespread depletion of myeloid lineage cells (which help prevent infection), this cell population can be so large (e.g., up to 100 billion neutrophils are produced daily in the human body) that efficacy can be compromised by poor distribution of suitable drugs and insufficient supply to achieve killing by T cells. Thus, targeting other cell populations such as leukemic blasts or even less immunosuppressive MDSCs may be hindered by the need for widespread distribution of the drug across CD33-positive normal myeloid cells and sufficient associated T cells to achieve killing. For these reasons, determining the appropriate dose, dosing schedule, and route of administration of T cell engagers is much more challenging than for monovalent, monospecific agents.

AMV564 Binding Properties and Dose Selection AMV564's bivalent design is reflected in its physical properties. AMV564 is highly potent, exhibiting cell killing with low receptor occupancy, and removal of CD33 target cells exhibiting picomolar or sub-picomolar EC50 values ex vivo or in vitro. AMV564 is a potent agonist and is capable of eliciting biological activity with low receptor occupancy or low target binding levels. Binding tests using flow cytometry showed that MDSC and leukemic blast cell line KG1 both strongly bound at 1 pM and 10 pM, whereas AMV564 at 1 pM or 10 pM bound neutrophils, polymorphonuclear (PMN) No leukocyte or monocyte binding is shown (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.

Therefore, if binding of target cells by AMV564 is sufficient for activity, but binding of other myeloid cells is minimal, both safety and antitumor (e.g., leukemic blasts) and/or antisuppression (MDSC) An optimal therapeutic window of cell activity may be obtained. Apart from safety considerations, excessive engagement of large cell populations encompassing normal myeloid lineages results in suboptimal drug distribution and an essential ternary complex that mimics the natural T cell synapse promoting cell killing. Efficacy may be reduced because there are insufficient T cells available to achieve body formation.

MDSC Depletion and Dose Selection Overcoming the suppressive tumor microenvironment is a major challenge in immunotherapy. A key cellular effector of the suppressive tumor microenvironment is MDSCs, which are associated with immune dysfunction, suppression of anti-tumor immunity, and refractoriness to immunotherapy. MDSCs suppress T cell and NK cell responses through various cytokines, active species, and pathways. Furthermore, they inhibit effective antigen presentation by dendritic cells in tumor-draining lymph nodes. In other aspects of the disclosure, AMV564 depletes low amounts of MDSCs in the periphery and bone marrow of AML patients. The rapid decline observed with low lead-in doses of AMV546 indicates strong binding and depletion of both monocytic and granulocytic MDSC populations. MDSCs are low in the periphery of healthy adults and are significantly elevated in cancer patients. However, nevertheless, MDSCs are generally relatively few cells compared to mature myeloid cells, and overall receptor occupancy is not favorable to the selectivity of bivalent T cell engagers such as AMV564. In some cases, their depletion and control effectiveness may be lower at higher doses.

Administering AMV564 to patients with solid tumors at doses of 5-50 μg resulting in a steady-state exposure in the range of approximately 0.1-5 pM results in favorable CD4 and CD8 T cell depletion that promotes MDSC depletion as well as restoration of anti-tumor immunity. It may be effective in promoting activation profiles and cytokine environments. Relatively high doses of 50-75 μg or 75-150 μg may also result in exposures that remain within the selective range for depleting MDSCs.

Circulating MDSCs are a pharmacodynamic biomarker of AMV564 and T cell responses. They are induced as a result of AMV564-stimulated T cell activation, as MDSCs are known to be induced by T cell activation. MDSCs reflect the binding of AMV564 to target cells (MDSCs) and the depletion of such cells, and in relation to the dosage of bivalent bispecific T cell engagers such as AMV564, the optimal Reflecting effective doses within the therapeutic index, these relatively rare cells can be effectively depleted compared to the remaining CD33-positive myeloid lineage.

Because AMV564 depletes MDSCs, treatment with AMV564, eg, according to the dosing regimens described herein, may be useful in depleting MDSCs in a variety of situations. For example, AMV564 can be used to deplete MDSCs in patients undergoing treatment with therapies that activate T cells or include administration of activated T cells.

Management of Cytokine Release Syndrome (CRS) CRS, which is not fully understood, involves activation of T cells and subsequent macrophage and secretion to produce and secrete IL-6, IL-1B, and other cytokines. It appears to be associated with activation of other bone marrow cells. CRS is commonly associated with T cell binding therapies such as T cell binding bispecific antibodies and CAR-T therapy. CRS is most evident at the beginning of administration. As shown below, for example, administering a dose of 15-50 μg of AMV564 via the subcutaneous route to solid tumor patients results in up to 10-40% increase in detectable peripheral interferon gamma (IFNγ) from baseline in the first dosing cycle. Robust T cell activation results as assessed by various metrics including fold increase. However, the increase in IL-6 was relatively modest (these two cytokines are approximately 1:1 or higher in IFΝγ) (see Figures 7A-7E), and IL-1B is not detected at any significant level. This favorable profile is consistent with the absence of CRS observed in patients in this clinical trial. This favorable profile of apparently strong T-cell activation with lack of CRS is due to depletion of MDSCs (which are capable of producing inflammatory cytokines), bivalent T-cell binding by AMV564 (more natural T-cell receptor binding) and lymphatic delivery and distribution kinetics associated with subcutaneous injection of AMV564. AMV564 has a good therapeutic index suitable for long-term administration, and these properties should also help alleviate CRS after completing lead-in administration to the target dose.

Combination Therapy Effective treatment of tumorigenesis is often achieved by combination therapy. The functional therapeutic index of AMV564, with a dose range that maximizes both efficacy and safety (particularly the lack of significant depletion of normal bone marrow cells), lends itself to a combination therapy approach. Combination strategies include checkpoint blockade in solid tumors (PD-1 or PDL-1 blockers), T cell activators and proliferators such as the cytokines IL-2, IL-10, and IL-15, checkpoint blockade (PD-1 or PDL-1 blockers), dual-targeting agents, such as targeting PD-1 or PDL-1), and immunosuppression (e.g., TGFβ), CAR therapy (expressed on T cells or NK cells), NK activation therapy, or Standard of care includes, but is not limited to, chemotherapy. In AML and MDS, the therapies listed above, along with other drugs established in AML, include, for example, hypomethylating agents (e.g. azacytidine, decitabine), differentiating agents (e.g. targeting IDH1/2), targeting It can also be used in combination with anti-apoptotic agents (eg, against FLT3), agents that target anti-apoptotic proteins such as BCL2 (eg, venetoclax), BCL-XL, or MCL1, or lenalidomide.

AMV564 is used to treat melanoma (e.g., unresectable or metastatic melanoma, patients with melanoma with lymph node metastases after complete resection), non-small cell lung cancer (NSCLC) (e.g., metastatic non-squamous NSCLC, III stage NSCLC, metastatic NSCLC expressing PD-L1), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), (e.g. locally advanced or metastatic urothelial cancer expressing PD-L1), frequently microsatellite unstable cancers (e.g. unresectable or metastatic, frequently microsatellite unstable (MSI- H) or mismatch repair deficiency), solid tumors that have progressed after previous treatment, breast cancer, uterine cancer, gastric cancer (e.g., recurrent locally advanced or metastatic gastric adenocarcinoma expressing PD-L1 or gastroesophageal cancer) It can be used alone or in combination to treat cervical cancer (junctional adenocarcinoma), cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), and renal cell carcinoma (RCC).

Figures 1A-1G show data demonstrating that AMV264 depletes MDSCs and activates T cells ex vivo, and that AMV564 binds to MSCs and KG-1 cells at 1 and 10 pm, but not to monocytes at these concentrations. Figure 1A shows that treatment of PBMCs with the CD33 ligand S100A9 results in proliferation of MDSCs and increased CD33 expression. Figures 1B-1E show that exposure to AMV564 counteracts reactive oxygen species (ROS) produced in response to S100A9 stimulation of PBMCs to proliferate MDSCs (Figure 1B), leading to selective depletion of MDSCs (Figure 1C), and increases the number and activation status (assessed by IFNγ positive fraction) of CD8 T cells (Figure 1D) and CD4 T cells (Figure 1E). See description of FIG. 1A. See legend to FIG. 1A. See description of FIG. 1A. See description of FIG. 1A. See description of FIG. 1A. See description of FIG. 1A. Figures 2A-2F show that AMV564 treatment results in depletion of peripheral blood and bone marrow MDSCs and AML blasts, without depletion of neutrophils. Figures 2A and 2B show depletion of MDSCs in peripheral blood. Figure 2C shows depletion of MDSCs in the bone marrow. Figures 2E-2G show the effects of AMV564 treatment on peripheral blood T cells (Figure 2E), peripheral blood neutrophils (Figure 2F), and peripheral blood blasts (Figure 2G). Light bars indicate lead-in dose days and dark bars indicate target dose days. See description of FIG. 2A. See description of FIG. 2A. See description of FIG. 2A. See description of FIG. 2A. See description of FIG. 2A. Figures 3A-3C provide data showing the effect of AM564 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 bars indicate lead-in dose days and dark bars indicate target dose days. Figures 4A-4D are data showing that AMV564 is a selective and potent conditional agonist. Single open circles and triangles indicate the results of CD3/CD28 stimulation in the absence of AMV564. Figure 4A shows that AMV564 induces potent dose-dependent cell death of KG1 with maximal levels similar to CD3-CD28 stimulation (Figure 4A). Figure 4B shows an increase in daughter cells reflecting T cell proliferation at levels comparable to or exceeding the CD3-CD28 reference stimulation. Figure 4C shows that there is no evidence that AMV564 promotes significant cell death on autologous monocytes or neutrophils. Figure 4D shows that unlike general T cell stimulation with CD3-CD28, there was no evidence of any induction of T cell proliferation by these cell populations. Figures 5A-5E are data showing that MDSC regulation is associated with Treg regulation in solid tumor patients. Figures 5A-5D have solid tumors and AMV564 (Figure 5A: ovary - 15 μg of AMV564; Figure 5B: skin 50 μg of AMV564; Figure 5C: small intestine - 15 μg of AMV564; Figure 5D: gastroesophageal junction - 15 μg of AMV564; We show that M-MDSC and G-MDSC are controlled in patients treated with AMV564 (once daily subcutaneous injection on days 1-5 and 8-12 of a 21-day cycle). The black squares are G-MDSCs and the black circles are M-MDSCs. Figure 5E shows the change in Tregs from baseline (B) over two treatment cycles (C1 and C2). The bar on the axis indicates the date of administration of AMV564. See description of FIG. 5A. See description of FIG. 5A. See description of FIG. 5A. See description of FIG. 5A. Figures 6A-6G are data showing improved CD8:Treg ratios with AMV564 therapy in solid tumor patients. Specifically, Figures 6A-6G show that AMV564 therapy (once daily subcutaneous injections on days 1-5 and 8-12 of a 21-day cycle (Figure 6A: Small intestine - stable (15 μg dose) ); Figure 6B: Ovarian - complete remission (15μg dose); Figure 6C: GE junction - progression (15μg dose); Figure 6D: Endometrium - stable (50μg dose); Figure 6E: Colon - progression ( Figure 6F: Skin-stable (50μg dose); Figure 6G: Appendiceal-stable (50μg dose)) shows that an increase in the CD8/Treg ratio was observed in most solid tumor patients. The dotted line shows the baseline ratio, the bar along the x-axis shows the date of administration, and the wide bar shows the ratio for healthy controls whose peripheral blood samples were processed in a similar flow-based assay. See description of FIG. 6A. See description of FIG. 6A. See description of FIG. 6A. See description of FIG. 6A. See description of FIG. 6A. See description of FIG. 6A. Figures 7A-7E show that AMV564 favors CD4 in ovarian cancer patients who received 15 μg of AMV564 (once daily subcutaneous injection on days 1-5 and 8-12 of a 21-day cycle). and CD8 promotes T cell polarization. Figure 7A shows the CD8/Treg ratio over 150 days. Figure 7B shows maintained or increased effector CD8 (TBX21 and/or granzyme B positive) and dynamic regulation of PD1 positive CD8 fraction. Figure 7C shows the dynamic increase in TBX21-positive CD4 helper T cells. Figure 7D shows continued increase in T cells. Figure 7E shows an increase in the percentage of CD8 T cells. Dotted lines indicate baseline proportions, bars along the x-axis indicate date of administration, and wide bars indicate proportions for healthy controls. See description of FIG. 7A. See description of FIG. 7A. See description of FIG. 7A. See description of FIG. 7A. Figures 8A-8F show IFΝγ cycle 1, IFΝγ cycle 2, IL-6 cycle 1, and IL-6 cycle 2 levels in six solid tumor patients treated with AMV564 (patient 1 (Figure 8A); patient 2 (FIG. 8B); Patient 3 (FIG. 8C); Patient 11 (FIG. 8D); Patient 9 (FIG. 8E); Patient 14 (FIG. 8F - cycle 1 only)). See description of FIG. 8A. See description of FIG. 8A. See description of FIG. 8A. See description of FIG. 8A. See description of FIG. 8A. Figures 9A-9D show measurements of M-MDSC and G-MDSC in four solid tumor patients treated with AMV564 in combination with pembrolizumab. Figures 9A and 9B show that 5 μg/kg/day, respectively, on days 1-5 and 8-12 of a 21-day cycle in combination with 200 mg pembrolizumab administered intravenously every 3 weeks (Q3W). Figure 2 shows observations of patients with solid tumors treated by subcutaneous injection of AMV564 once daily. Figures 9C and 9D show 15 μg/day on days 1-5 and 8-12 of a 21-day cycle, respectively, in combination with 200 mg pembrolizumab administered intravenously every 3 weeks (Q3W). Figure 1 shows observations of solid tumor patients treated by subcutaneous injection of AMV564 once daily. The black squares are G-MDSCs and the black circles are M-MDSCs. The bar on the axis indicates the date of administration of AMV564. See description of FIG. 9A. See description of FIG. 9A. See description of FIG. 9A. Figures 10A-D show the effect of AMV564 in combination with pembrolizumab on T-Bet and granzyme B positive CD8 cells and CD8/Treg ratios in two solid tumor patients. Figures 10A and 10C show AMV564 (15 μg subcutaneously once daily on days 1-5 and 8-12, respectively, of a 21-day cycle) in combination with pembrolizumab (200 mg intravenously Q3W). Results are shown for patient 15 treated with injection). Figures 10B and 10D show AMV564 (15 μg subcutaneous injection once daily on days 1-5 and 8-12 of a 21-day cycle) in combination with pembrolizumab (200 mg intravenously Q3W). Results for patient 16 treated with ) are shown. The dotted line shows the baseline ratio, the bar along the x-axis shows the date of administration of AMV564, and the wide bar shows the ratio for healthy controls. Figures 11A-11B show the effect of AMV564 treatment in combination with pembrolizumab on CD8 cell proliferation and activation in two solid tumor patients. Figures 11A and 11B show AMV564 (15 μg subcutaneous injection once daily on days 1-5 and 8-12 of a 21-day cycle) in combination with pembrolizumab (200 mg intravenously Q3W). The data observed for patient 15 (FIG. 11A) and patient 16 (FIG. 11B) treated with ) are shown. Figures 12A-12B show the effect of AMV564 on the levels of M-MDSC and G-MDSC cells over the course of five treatment cycles (*p<0.05, **p<0.01). Figure 12A shows the effect on M-MDSC levels of treating solid tumor patients with subcutaneous administration of 15 or 50 μg of AMV564. Figure 12B shows the effect on G-MDSC levels of treating solid tumor patients with subcutaneous administration of 15 or 50 μg of AMV564. Figures 13A-13B show the effects of AMV564 alone or in combination with pembrolizumab on the co-expression of granzyme B and TBX21 in CD8+ T cells (Figure 13A) and the frequency of granzyme B+ CD8+ cells (Figure 13). Figures 14A-14B show the effect of AMV564 alone or in combination with pembrolizumab on the levels of IFΝγ and IL-6. Figure 14A shows IFΝγ and IL-6 levels observed in solid tumor patients treated with 15 or 50 μg of AMV564 alone or in combination with pembrolizumab (n=11 monotherapy, n=4 combination). Figure 14B shows that compared to other T cell engagers, AMV564 exhibited a favorable IFΝγ to IL-6 ratio of approximately 1:1. See description of FIG. 14A. Figures 15A-15C show the effects of AMV564 alone or in combination with pembrolizumab on various cytokine levels in patients and in vitro cytotoxicity assays. Figure 15A shows the effect of AMV564 alone or in combination with pembrolizumab on the levels of TNFα, IL-1β, and IL-10 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 an AMV564 cytotoxicity assay performed using KG-1 cells as target cells. This is a continuation of FIG. 15A-1. See description of FIG. 15A-1. See description of FIG. 15A-1. Figures 16A-16C show the effects of AMV564 alone or in combination with pembrolizumab on T cell repertoires in three different patients. Figure 16A shows the expansion of the T cell repertoire of patients with small bowel cancer. Figure 16B shows expansion of the T cell repertoire of patients with penile squamous cell carcinoma. Figure 16C shows expansion of the T cell repertoire of patients with pancreatic cancer. Orange circles indicate clones that were significantly expanded or undetectable as baseline. [0023] Figures 17A-17C show the effect of AMV564 on CD8 and CD8 memory cells and T cell reconstitution in ovarian cancer patients with confirmed RECIST CR. Figure 17A shows the increase in CD8 cells over the course of treatment in an ovarian cancer patient treated with 15 μg AMV564. Figure 17B shows the increase in CD8 memory cells over the course of treatment in an ovarian cancer patient treated with 15 μg AMV564. Figure 17C shows tracking of specific T cell reconstitution over treatment time points in an ovarian cancer patient. See description of FIG. 17A. See description of FIG. 17A.

Detailed description AMV564
AMV564 is a homodimer of SEQ ID NO:1. AMV564 is described in US9212225 (Diabody 16, SEQ ID NO: 113 without the 6His tag at the amino terminus) and WO2016/196230 (SEQ ID NO: 139). The pharmaceutical composition of AMV564 comprises a homodimer of a polypeptide having the amino acid sequence of SEQ ID NO: 1 and a pharmaceutically acceptable carrier or excipient.

AMV564

Example 1: AMV564 depletes MDSCs and activates T cells ex vivo In this study, we found that ex vivo AMV564 treatment of primary cells (PBMCs, MDS bone marrow, tumor PBMCs) not only depletes MDSCs but also activates T cells. Figure 1A shows that treatment of PBMCs with CD33 ligand S100A9 leads to MDSC proliferation and increased CD33 expression. Exposure to AMV564 counteracts reactive oxygen species (ROS) produced in response to S100A9 stimulation of PBMCs to proliferate MDSCs (Figure 1B), leading to selective depletion of MDSCs (Figure 1C) and increased numbers and activation status (assessed by IFNγ positive fraction) of CD8 T cells (Figure 1D) and CD4 T cells (Figure 1E).

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

AMV564 binds to MDSCs (and leukemic blast cell line KG1) at 1 and 10 pM (FIG. 1F). However, there is virtually no evidence of binding to monocytes, neutrophils, and polymorphonuclear leukocytes (PMNs) at these concentrations. These concentrations are within the range of exposure observed for administration of AMV564 by the subcutaneous route at doses of 5-15-50 μg (approximately 0.1-5 pM). As shown in FIG. 1G.

Example 2: AMV564 depletes MDSCs and activates T cells in patients In this clinical trial, AMV564 treatment resulted in the depletion of peripheral blood and bone marrow MDSCs and AML blasts without depletion of neutrophils. It was found that this resulted in depletion of (FIGS. 2A-2F). Rapid depletion of both monocytic and granulocytic MDSCs is evident with little or no effect on circulating neutrophil or monocyte populations. Evidence of early T cell activation is evident by a rapid redistribution/marginal trend of T cells (this apparent transient lymphopenia is associated with T cell activation and migration to lymph nodes and tissues). ). In Figures 2A-2F, the period of lead-in AMV564 dosing (15 μg, 3 days) is shown as a lighter colored bar, and the target AMV564 dose (100 μg) is shown as a darker colored bar. MDSC depletion was observed in peripheral blood (Figures 2A and 2B) and bone marrow (Figure 2C). Figures 2D-2E show the effects of AMV564 treatment on peripheral blood T cells, peripheral blood neutrophils, and peripheral blood blasts, respectively.

Example 3: AMV564 depletes MDSCs in patients with solid tumors Upon lead-in administration (1-3 days in some patients), an initial increase in peripheral blood MDSCs in response to T cell activation was observed ( Figures 3A-C). Consistent with T cell activation, a rapid redistribution/marginal trend of peripheral blood T cells 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 significantly reduced compared to baseline when assessed on day 15. However, upon discontinuation of AMV564 treatment, both bone marrow and peripheral blood MDSCs returned.

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

Example 5: Phase 1 clinical trial of AMV564 in patients with solid tumors. Adult patients with . Patients had an ECOG performance status of 2 or less and adequate organ function. Patients were treated with AMV564 alone (15, 50, or 75 μg/day) or with AMV564 (5, 15, or 50 μg/day) in combination with 200 mg intravenous pembrolizumab every 3 weeks (Q3W). . In all cases, AMV564 was administered once daily by subcutaneous injection on days 1-5 and 8-12 of a 21-day cycle. AMV564 was well tolerated and pharmacodynamic analysis showed reduced immunosuppression (decreased MDSC and Tregs) in this phase 1 population of heterogeneous cancer patients previously treated with other therapies. as well as evidence of promotion of effector CD8 and Th1 CD4 responses.

In general, patients treated with AMV564 demonstrated a cytokine profile (increased IFNγ, IL-15, IL-18, soluble granzyme B, and CXCL10) consistent with activation of T cells, including CD4 Th1 helper cells, antigen-presenting cells, and improved trafficking of T cells to tissues, including tumor tissue. Although robust T cell activation was observed, there was no development of cytokine release syndrome.

Example 6: MDSC regulation is associated with Treg regulation in patients with solid tumors. AMV564; Figure 5D: Gastroesophageal Junction - 15 μg of AMV564) in patients treated (once daily subcutaneous injection on days 1-5 and 8-12 of a 21-day cycle). and G-MDSC are controlled. Figure 5E shows the change in Tregs from baseline (B) over two treatment cycles (C1 and C2).

Example 7: AMV564 therapy improves CD8:Treg ratio in patients with solid tumors. subcutaneous injection of (Figure 6A: small intestine - stable (15μg dose); Figure 6B: ovary - complete remission (15μg dose); Figure 6C: GE junction - progression (15μg dose); Figure 6D: endometrium - Stable (50 μg dose), Figure 6E: Colon - Progressive (50 μg dose), Figure 6F: Skin - stable (50 μg dose), Figure 6G: Appendix - stable (50 μg dose)) in solid tumor patients. It shows that an increase in CD8/Treg ratio was observed in most subjects.

Example 8: AMV564 promotes favorable CD4 and CD8 T cell polarization in ovarian cancer patients Platinum-based chemotherapy, surgery, radiation, pembrolizumab (best stable response, completed 6 months before study initiation), and Ovarian cancer patients who had received multiple prior therapies with niraparib and letrozole received 15 μg of AMV564 (once daily subcutaneous injection on days 1-5 and 8-12 of a 21-day cycle). ). This patient showed a continued increase in CD8/Treg, an increase in CD8%, maintenance or increase in effector CD8 (TBX21 and/or granzyme B positive) and memory CD8 cells, a dynamic increase in TBX21 positive CD4 helper T cells and PD1 showed a dynamic regulation of the positive CD8 fraction, but no significant overall increase as shown in Figures 7A-7E. The patient progressed from stable to partial remission to complete remission as assessed by CT scan at 6-8 week intervals.

Example 9: Patients treated with AMV564 show signs of T cell activation without CRS. The results of measurements of IFΝγ and IL-6 in 6 solid tumor patients treated with subcutaneous injection (once daily subcutaneous injection on days 1 to 5 and days 8 to 12) are shown. Taken together, there is clear evidence of systemic IFΝγ production without excessive IL-6 production (IFΝγ:IL-6 ratio is approximately 1:1 or better in most patients). It was good).

Over five treatment cycles, solid tumor patients (data not shown) showed increased levels of MHC class I upregulation, dendritic cell activation, and Consistent with T cell trafficking, there was an increase in IFΝγ, TNFα, and IL18 as well as other factors (decreased MDSC-inducing factor G-CSF). This same patient entered the study with low CD8/Treg at baseline. Improvements were observed throughout treatment, particularly around cycle 3, where CD8 T cell proliferation and activation (Ki67 and CD38 fraction) was observed. This reflects the timing at which increases in cytokines and factors are observed, coinciding with dendritic cell activation and improved Th1 responses, and suggests that AMV564 may be more favorable over time, even in this late stage patient. This suggests that it promoted immune polarization.

Example 10: Treatment with AMV564 and Pembrolizumab Figures 9A-9D are shown on days 1-5 and 8-5 of a 21-day cycle in combination with intravenous administration of 200 mg pembrolizumab every three weeks (Q3W). M-MDSC and The results of G-MDSC measurement are shown. AMV564 administration dates are indicated by bars along the x-axis, and pembrolizumab treatment dates are indicated by asterisk symbols. As can be seen, very good MDSC control was observed.

Figures 10A-10D show AMV564 (15 μg subcutaneously once daily on days 1-5 and 8-12 of a 21-day cycle) in combination with pembrolizumab (200 mg intravenously Q3W). Data obtained from two treated patients (FIGS. 10A and 10C: patient 15, and FIGS. 10B and 10D: patient 16) are shown. This data shows evidence of a significant increase in the CD8 effector cell fraction and a significant increase in the CD8/Treg ratio in cycles 1-2. The data also show proliferation of T-Bet and granzyme B positive CD8 cells. These effects were not evident in the 5 μg AMV564 combination cohort in this study.

Figures 11A-11B show AMV564 (15 μg subcutaneously once daily on days 1-5 and 8-12 of a 21-day cycle) in combination with pembrolizumab (200 mg intravenously Q3W). CD8 T cell proliferation data obtained from two treated patients (FIG. 11A: patient 15 and FIG. 11B: patient 16) is shown. This data shows evidence of a significant increase in CD8 proliferation (as assessed by CD8 Ki67) and activation (as assessed by CD8 CD38). Two out of three patients treated with AMV564 and pembrolizumab experienced a significant and rapid increase from low baseline levels, suggesting a potential benefit from the combination. There is.

Example 11: In solid tumor patients, AMV564 selectively targets M-MDSC and G-MDSC for depletion and activates T cells Solid tumor patients treated with subcutaneous administration of 15 or 50 μg of AMV564 M-MDSC and G-MDSC were measured. As can be seen in Figures 12A-12B, treatment was accompanied by a decrease in both MDSC subtypes. This is important because increases in M-MDSCs often correlate with decreased levels of peripheral T cells.

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

Example 12: AMV564 induces a modulatory immune response Solid tumor patients treated with 15 or 50 μg of AMV564 alone or in combination with pembrolizumab (n=11 monotherapy, n=4 in combination) showed IFΝγ versus IL- 6 showed a good ratio of about 1:1 (FIGS. 14A and 14B). In many cases, treatment with other T cell engagers results in a ratio of 0.1 to 0.01.

Levels of IL-6, IL-1β, IL-10, and TNFα, all bone marrow-derived cytokines, remained low in solid tumor patients treated with AMV564 (FIGS. 15A and 15B, data not shown). In contrast, these patients had elevated levels of proinflammatory cytokines that promote Th1 polarization, macrophage activation, and T cell trafficking to tumors (Figures 15A and 15B, data not shown). ).

In vitro cytotoxicity assays using KG-1 cells as target cells showed that AMV564 was associated with a good IFΝγ to IL-6 ratio over a wide range of AMV564 ratios (FIG. 15C).

Example 13: AMV564 Expands the Peripheral T Cell Repertoire The T cell repertoire of three patients (small intestine cancer, penile squamous cell carcinoma, and pancreatic cancer) was expanded during different treatment cycles (day 1 of cycle 1). and comparison with day 1 of cycle 2) and evaluated by deep sequencing of TCRβ CDR3. To correlate the effect of treatment on TCR repertoire and disease progression, clones that were expanded, suppressed, or generated de novo during treatment were evaluated. 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). Approximately 30-300 differentially detected T cell clones were observed per patient, some of which were undetectable or very low at baseline.

Example 14: T-cell repertoire expansion in ovarian cancer patients was associated with increased CD8 memory cells In ovarian cancer patients treated with 15 μg AMV564 and confirmed RECIST CR, CD8 cells ( Figure 17A) and CD8 memory cells (Figure 17B) appeared increased.

Following the reconstitution of specific T cells over treatment time points, we found that several clones proliferated and eventually dominated this patient's repertoire (FIG. 17C). Two of the eight most proliferated T cell clones correspond to CDR3 sequences consistent with T cell targeting SLC3A2 neoantigen (upregulated in some cancers and often associated with poor prognosis) (Fig. 17C).

INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned herein are incorporated by reference as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. , herein incorporated by reference in its entirety. 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 appended claims.

Claims (41)

A method for treating a patient suffering from a solid tumor, the method comprising administering immunotherapy to the patient and administering AMV564, wherein the AMV564 is administered before, after, or in conjunction with the immunotherapy. The method described above. 2. The method of claim 1, wherein the AMV564 is administered by subcutaneous injection. The method of claim 1, wherein the AMV564 is administered within 4 to 6 weeks after the immunotherapy is administered. 4. The method of claim 3, wherein the AMV564 is administered on at least 7 days during a 14 day period. 5. The method of claim 4, wherein the AMV6564 is administered 10 days during a 14 day period. 6. The method of claim 5, wherein the AMV6564 is administered twice for 5 consecutive days within a 14 day period. 6. The method of claim 5, wherein the AMV6564 is administered for 5 consecutive days, then 2 days without administration, and then administered for 5 consecutive days. 8. The method of any one of claims 4 to 7, wherein the AMV564 is administered in a 21 day cycle, and is administered on at least 7 days during a 14 day period of the 21 day cycle, and then not administered for 7 days. . 9. The method of claim 8, wherein the 21 day cycle is repeated at least twice. 10. The method of claim 9, wherein the AMV564 is administered for at least 10 days during a 14 day period, wherein the AMV564 is administered for 5 consecutive days, then 2 days without administration, and then 5 consecutive days. . 11. The dose of AMV564 administered is 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 μg for each day administered. the method of. 2. The method of claim 1, wherein the AMV564 is administered at 15 [mu]g/day for 5 days during the first week of treatment, and then at 50 [mu]g once a week. 2. The method of claim 1, wherein the AMV564 is administered at 5-50 μg once a week. The method of claim 1, wherein the AMV564 is administered at 15 μg/day for 5 days during the first week of treatment, and then once a week at 15 μg. The method of claim 1, wherein the AMV564 is administered at 15 μg/day for 5 days during the first week of treatment, and then once a week at 15 μg to 50 μg. 16. 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 solid tumor is breast-pancreatic cancer, ovarian cancer, colon cancer, rectal cancer, non-small cell lung cancer, urothelial cancer, squamous cell cancer, rectal cancer, penile cancer, or endometrial cancer. The method according to claim 1, wherein the method is selected from cancer, small intestine cancer, and appendiceal cancer. 2. The method of claim 1, wherein the administration of AMV564 achieves a constant exposure of 0.1-5 pM AMV564. 2. The method of claim 1, wherein the administration of AMV564 achieves a constant exposure of 0.5-3 pM AMV564. 2. The method of claim 1, wherein the administration of AMV564 achieves a constant exposure of 1-5 pM AMV564. 2. The method of claim 1, wherein the immunotherapy is an anti-PD-L1 antibody or an anti-PD-1 antibody. 22. The method of claim 21, wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, or cemiplimab. 22. The method of claim 21, wherein the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab. 2. The immunotherapy of claim 1, wherein the immunotherapy is CAR T cell therapy and the AMV564 is administered 1 to 5 days, 5 to 10 days, or 5 to 14 days after the CAR T cell therapy is administered. Method. A method for treating cancer in a patient, comprising administering AMV564 to the patient, wherein the cancer does not express CD33. 26. The method of claim 25, wherein the AMV564 is administered by subcutaneous injection. 27. The method of claim 25 or 26, wherein the AMV564 is administered at 15 [mu]g/day for 5 days during the first week of treatment, and then once a week at 50 [mu]g. 26. The method of claim 25, wherein the AMV564 is administered at 50 μg once a week. 26. The method of claim 25, wherein the AMV564 is administered at 15 [mu]g/day for 5 days during the first week of treatment, and then once a week at 15 [mu]g. 26. The method of claim 25, wherein the AMV564 is administered at 15 μg/day for 5 days during the first week of treatment, and then once a week at 15 μg to 50 μg. The method of claim 25, wherein the dose of AMV564 injected is 5 to 50 μg. The solid tumor is pancreatic cancer, ovarian cancer, colon cancer, rectal cancer, non-small cell lung cancer, urothelial cancer, squamous cell cancer, rectal cancer, penile cancer, endometrial cancer. , small intestine cancer, and appendiceal cancer. The solid tumor is small cell lung cancer (NSCLC) (e.g., metastatic non-squamous NSCLC, stage III NSCLC, metastatic NSCLC expressing PD-L1), melanoma, Merkel cell, frequent microsatellite instability cancer. (e.g., unresectable or metastatic, microsatellite instability-high (MSI-H) or mismatch repair deficiency) and the patient is undergoing checkpoint blockade. The method described in section. The patient has melanoma (e.g., unresectable or metastatic melanoma, melanoma with lymph node metastases after complete resection), non-small cell lung cancer (NSCLC) (e.g., metastatic non-squamous NSCLC, stage III NSCLC, metastatic NSCLC expressing PD-L1), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelium cancer (e.g., locally advanced or metastatic urothelial cancer that expresses PD-L1), microsatellite instability cancer (e.g., unresectable or metastatic, microsatellite instability (MSI) -H) or mismatch repair deficiency), solid tumors that have progressed after previous treatment, gastric cancer (e.g., recurrent locally advanced or metastatic gastric adenocarcinoma or gastroesophageal junction adenocarcinoma expressing PD-L1); ), cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), and renal cell carcinoma (RCC). The method described in. A method for expanding T cells, said method comprising culturing said T cells in the presence of AMV564. 36. The method of claim 35, wherein the T cell expresses a chimeric antigen receptor. 37. The method of claim 35 or 36, wherein said culturing lasts for at least 5 days. A method for expanding NK cells, the method comprising culturing the NK cells in the presence of AMV564. 39. The method of claim 38, wherein the NK cell expresses a chimeric antigen receptor. 40. The method of claim 38 or 39, wherein said culturing lasts for at least 5 days. A method for expanding cytotoxic lymphocytes (CTL), said method comprising culturing said CTL in the presence of AMV564.

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